Cyanobacteria having improved photosynthetic activity

ABSTRACT

This disclosure describes modified photosynthetic microorganisms, including Cyanobacteria, that have a reduced amount of a light harvesting protein (LHP) and contain one or more introduced or overexpressed polynucleotides encoding one or more enzymes associated with lipid biosynthesis, and which are capable of producing increased amounts of fatty acids and/or synthesizing triglycerides.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation of U.S. Non-Provisional patentapplication Ser. No. 15/879,993 filed on Jan. 25, 2018, now U.S. Pat.No. 10,563,168, which is a continuation of U.S. Non-Provisional patentapplication Ser. No. 14/775,606 filed on Sep. 11, 2015, now U.S. Pat.No. 9,914,907, which is a National Stage Entry of PCT/US14/22842, filedon Mar. 10, 2014 which claims priority to U.S. Provisional PatentApplication No. 61,780,755 filed on Mar. 13, 2013, entitled“Cyanobacteria Having Improved Photosynthetic Activity,” which is herebyincorporated by reference in their entirety.

SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is “LUBI-011_03US_SeqList_ST25.txt.” The text fileis about 734 KB, was created on Feb. 14, 2020, and is being submittedelectronically via EFS-Web.

BACKGROUND

Certain organisms can be utilized as a source of oil such astriglycerides in the production of biofuels. For example, algaenaturally produce triglycerides as energy storage molecules, and certainbiofuel-related technologies are presently focused on the use of algaeas a feedstock for biofuels. Algae are photosynthetic organisms, and theuse of triglyceride-producing organisms such as algae provides theability to produce biodiesel from sunlight, water, CO₂, macronutrients,and micronutrients. Algae, however, cannot be readily geneticallymanipulated, and produce much less oil (i.e., triglycerides) underculture conditions than in the wild.

Like algae, Cyanobacteria obtain energy from photosynthesis, utilizingchlorophyll A and water to reduce CO₂. Certain Cyanobacteria can producemetabolites, such as carbohydrates, proteins, and fatty acids, from justsunlight, CO₂, water, and inorganic salts. Unlike algae, Cyanobacteriacan be genetically manipulated. For example, S. elongatus PCC 7942(hereafter referred to as “S. elongatus PCC 7942”) is a geneticallymanipulable, oligotrophic Cyanobacterium that thrives in low nutrientlevel conditions, and in the wild accumulates fatty acids in the form oflipid membranes to about 4 to 8% by dry weight. Cyanobacteria expresslight harvesting protein (LHP), which collects photons (i.e., lightenergy) and channel their energy to the photosynthetic reaction centers.However, although these proteins are extremely efficient at harvestinglight, their capacity to use light for photosynthesis is easilysaturated.

Clearly, therefore, there is a need in the art for modifiedphotosynthetic microorganisms, including Cyanobacteria, capable ofperforming improved photosynthetic activity and producing oil such astriglycerides, e.g., to be used as feedstock in the production ofbiofuels and/or various specialty chemicals.

BRIEF SUMMARY

Embodiments of the present invention relate to the demonstration thatphotosynthetic microorganisms, including Cyanobacteria, can be modifiedto reduce expression of light harvesting proteins and unexpectedlyincrease photosynthetic activity. The modified Cyanobacteria can becultured to produce carbon-containing compounds such as lipids andtriglycerides. In certain embodiments, the modified photosyntheticmicroorganisms, e.g., Cyanobacteria, of the present invention can alsocomprise one or more polynucleotides encoding one or more enzymesassociated with neutral lipid synthesis and lipid packaging protein.

In some embodiments, the present disclosure includes a cell culturecomprising modified Cyanobacteria that have a reduced amount of a lightharvesting protein (LHP), wherein, as compared to correspondingwild-type Cyanobacteria, the modified Cyanobacterium grow, divide orboth at an increased rate, and/or have an increased level ofphotosynthetic activity.

In some embodiments, the present disclosure includes a method forgenerating modified Cyanobacteria that comprises modifying one or morepolynucleotides associated with light harvesting proteins ofCyanobacteria to generate the modified Cyanobacteria, wherein themodified cyanobacteria have an increased level of photosyntheticactivity as compared to corresponding wild-type Cyanobacteria. In theseembodiments and other embodiments, the present disclosure include amethod for generating modified Cyanobacteria, comprising: culturingCyanobacteria under a stress condition; and isolating modifiedCyanobacteria that have an increased level of photosynthetic activity ascompared to corresponding wild-type Cyanobacteria, wherein the stresscondition comprises culturing under increased light, culturing inmetronidazole containing growth media or both.

In some embodiments, the present disclosure includes a modifiedCyanobacterium comprising a reduced amount of a light harvesting proteinas compared to a corresponding wild-type Cyanobacterium. In particularembodiments, the modified Cyanobacterium has reduced expression of oneor more genes of light harvesting protein biosynthesis or transportationpathway as compared to the corresponding wild-type Cyanobacterium.

In some embodiments, the present disclosure includes a method forproducing a carbon-containing compound, comprising: culturing modifiedCyanobacteria comprising a reduced amount of a light harvesting proteinas compared to a corresponding wild-type Cyanobacteria to therebyproduce a carbon-containing compound; and harvesting thecarbon-containing compound, wherein the modified cyanobacteria have anincreased level of photosynthetic activity as compared to thecorresponding wild-type Cyanobacteria.

In particular embodiments of the modified Cyanobacteria/Cyanobacteriumand the related cell culture as well as the methods, the modifiedCyanobacteria/Cyanobacterium contain modulations of the nblA, rpaB,pbsB, pbsC, or Phycobiliprotein gene, individually or in variouscombinations, may produce and accumulate significantly reduced levels ofLHP as compared to wild-type Cyanobacteria. In some instances, thephotosynthetic activity is measured based on at least one of a growthrate, a level of oxygen evolution, or a biomass accumulation rate.

BRIEF DESCRIPTION OF THE FIGURE

FIGS. 1A and 1B show a measurement of phycobilisomes, comparingwild-type to NbIA overexpressor. Synechococcus PCC7942 wild type,PCC7942 with an exogenous NbIA gene, uninduced or PCC7942 with anexogenous NbIA gene, induced. Samples were collected at time zero (FIG.1A) and at six hours (FIG. 1B) after induction of the NbIA gene. Wholecells were examined by spectrophotometry, and absorbance as a functionof wavelength was determined. The three major peaks represent absorptionby chlorophyll A (at approximately 420 and 680 nm) and byphycobiliprotein (at approximately 630 nm). Induction of NbIA caused arapid decrease in light absorption by phycobiliprotein. Some reductionin the 680 nm chlorophyll A peak was also observed. These data show thatphycobilisomes are reduced after modulated NbIA expression, indicatingthat modulating NbIA expression reduce phycobilisome abundance.

FIG. 2 shows normalized photosynthetic activities of suspensions of wildtype and modified cyanobacteria containing an arabinose-inducedover-expression system of the gene nblA. Triplicate cultures ofwild-type Synechococcus sp. PCC 7942, pBAD nblA uninduced and pBAD nblAinduced (with 0.02% arabinose added) were harvested in log-phase(between OD₇₅₀ values of 0.4 and 0.6) and re-suspended in BG-11 mediumwith 20 mM Potassium Phosphate (pH 7.5) and 10 mM Sodium Bicarbonateadditions. These suspensions were illuminated with Red+Blue LEDs in acalibrated Walz Dual Pam 100 Fluorometer (Walz, Germany) to total lightintensities between 0 and 600 μE*m⁻²*s⁻¹ and oxygen concentration wasmonitored by a NeoFox Oxygen Sensor (Ocean Optics, USA) continuouslyevery second for 120 seconds at each light intensity. The slopes of thelinear O₂ production rate was then found and plotted above for eachculture time (n=3 for each type).

FIG. 3 shows representative normalized whole-cell absorbance spectra ofwild type (WT), pBAD nblA uninduced (U), and pBAD nblA induced with0.02% arabinose (I). Suspensions measured for FIG. 1 were diluted 1:2 inBG-11 and measured in a spectrophotometer. Absorbances were normalizedby taking the absorbance value at each wavelength (A) and dividing bythe absorbance value at 800 nm (A800) and subtracting 1 from that ratio.

FIGS. 4A and 4B show growth of a control strain in the presence of 0.02%arabinose (which does not change optical characteristics in presence ofarabinose), pBAD nblA, and pBAD nblA in the presence of 0.02% arabinosein photobioreactors as monitored by Optical Density (FIG. 4A) and dryweight (FIG. 4B). Optical density was measured by taking aliquots ofculture and measuring absorbance at 750 nm. Dry weight was measured byweighing 0.2 micron pre-weight filters with dried cells from 5-10 mLculture aliquots.

FIG. 5 shows relative expression (log₂ fold change) of total nblAtranscripts for NY016 and NY001 (in the presence of 0.02% arabinose)versus wild-type as measured by q-RT-PCR. NY001 is a strain with anative copy of nblA gene behind its native promoter plus a second copyof nblA gene behind an arabinose inducible promoter (pBAD). NY016 is astrain with a native copy of the nblA gene behind its native promoterplus a second copy of nblA gene behind a constitutive high-expressionpromoter (pSYNPCC7942_1306).

FIG. 6 shows normalized photosynthetic activities of wild-type (WT),pBAD nblA induced with 0.02% arabinose (NY001_I), and a strainconstitutively overexpressing nblA (NY016) suspended in BG-11 mediumwith 20 mM Sodium Phosphate (pH 7.1) and 10 mM Sodium Bicarbonate addedat OD750 values between 0.2 and 2.0.

FIGS. 7A and 7B show growth of a control strain (NY048) and NY016 inphotobioreactors as monitored by Optical Density (FIG. 7A) and dryweight (FIG. 7B). Optical density was measured by taking aliquots ofculture and measuring absorbance at 750 nm. Dry weight was measured byweighing 0.2 micron pre-weight filters with dried cells from 5-10 mLculture aliquots.

FIG. 8 shows representative normalized whole-cell absorbance spectra ofcontrol strain (NY048) and strain with constitutive overexpression ofnblA (NY016). Absorbances were normalized by taking the absorbance valueat each wavelength (A) and dividing by the absorbance value at 800 nm(A800) and subtracting 1 from that ratio.

FIG. 9 shows High Light Kill Curve using WT and High Light (HL) mutantsDC1, DC3 and DC4. Cultures of these strains were cultured and diluted to0.1 OD₇₅=1.0*10⁷ cells/mL (Colony forming units (CFU's)/mL shown as“prelight”). Samples were then exposed to >3000 μE*m⁻²*s⁻¹ white LEDlight for 3 exposures (rounds 1-3) each for 1 hour. After each round,culture aliquots were plated and CFUs were counted from cultured plates˜1 week later. WT has the strongest decrease in cell density followingtreatment rounds.

FIG. 10 shows normalized spectra of high-light resistant mutants (HLmutants) DC1 and DC4 relative to WT. Absorbances were normalized bytaking the absorbance value at each wavelength (A) and dividing by theabsorbance value at 800 nm (A800) and subtracting 1 from that ratio.

FIG. 11 shows photosynthetic activities as measured by oxygen evolutionfor high-light mutants DC1 and DC4 versus WT.

FIG. 12 shows candidate metronidazole (MZ) resistant mutants. Culturesof the wild type and metronidazole (MZ) resistant mutants were incubatedwith 4 mM MZ for the times indicated on the left side. Five μL ofculture (˜5000 cells) were spotted from each flask and grown on BG-11plate at a light intensity of 100 μE*m⁻²*s⁻¹.

FIG. 13 shows a plot of OD₇₅₀ values of NY0056 grown in BG11+20 mMNaPi+0, 5, 20, 40, 60, 80, 100, or 120 uMisopropyl-β-D-1-thiogalactopyranoside (IPTG) (singlet cultures).

FIG. 14 shows a spectra of these cultures from FIG. 13 normalized toA800.

FIG. 15 shows plotted O₂ evolution curves of the cultures from FIG. 13in the WALZ dual PAM 100 after ˜30 hours growth for cells resuspended at˜2.0 OD750 in BG11+20 mM NaPi+10 mM NaHCO₃.

FIG. 16 shows a plot of the maximum light O₂ evolution for each cultureof FIG. 15 at varying IPTG.

FIG. 17 shows the A800-normalized spectra of cultures grown for 30+ hrsin BG11+20 mM NaPi+0, 5, 10, 20, and 30 uM IPTG with NY056 in BG11+20 mMNaPi+0, 5, 10, 20, and 30 uM IPTG (singlet cultures).

FIG. 18 shows shows plotted O₂ evolution curves of the cultures fromFIG. 17 in the WALZ dual PAM 100 after ˜30 hours growth for cellsresuspended at ˜2.0 OD750 in BG11+20 mM NaPi+10 mM NaHCO₃.

FIG. 19 shows a plot of the maximum light O₂ evolution for each cultureof FIG. 18 at varying IPTG.

FIG. 20 shows the maximum light O₂ for both experiments (FIGS. 13 and17) combined in one maximum light O₂ graph.

DETAILED DESCRIPTION

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, exemplary methods andmaterials are described. For the purposes of the present invention, thefollowing terms are defined below.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

By “about” is meant a quantity, level, value, number, frequency,percentage, dimension, size, amount, weight or length that varies by asmuch as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a referencequantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length.

The term “biologically active fragment”, as applied to fragments of areference polynucleotide or polypeptide sequence, refers to a fragmentthat has at least about 0.1, 0.5, 1, 2, 5, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96,97, 98, 99, 100, 110, 120, 150, 200, 300, 400, 500, 600, 700, 800, 900,1000% or more of the activity of a reference sequence. The term“reference sequence” refers generally to a nucleic acid coding sequence,or amino acid sequence, of any enzyme having a diacylglycerolacyltransferase activity, a phosphatidate phosphatase activity, and/oran acetyl-CoA carboxylase activity, as described herein (see, e.g., SEQID NOS:1-9).

Included within the scope of the present invention are biologicallyactive fragments of at least about 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200,220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 500, 600 or morecontiguous nucleotides or amino acid residues in length, including allintegers in between, which comprise or encode a polypeptide having anenzymatic activity of a reference polynucleotide or polypeptide.Representative biologically active fragments generally participate in aninteraction, e.g., an intra-molecular or an inter-molecular interaction.An inter-molecular interaction can be a specific binding interaction oran enzymatic interaction. Examples of enzymatic interactions or activityinclude diacylglycerol acyltransferase activity, phosphatidatephosphatase activity, and/or acetyl-CoA carboxylase activity, asdescribed herein.

By “coding sequence” is meant any nucleic acid sequence that contributesto the code for the polypeptide product of a gene. By contrast, the term“non-coding sequence” refers to any nucleic acid sequence that does notcontribute to the code for the polypeptide product of a gene.

Throughout this specification, unless the context requires otherwise,the words “comprise”, “comprises” and “comprising” will be understood toimply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements.

By “consisting of” is meant including, and limited to, whatever followsthe phrase “consisting of.” Thus, the phrase “consisting of” indicatesthat the listed elements are required or mandatory, and that no otherelements may be present.

By “consisting essentially of” Is meant including any elements listedafter the phrase, and limited to other elements that do not interferewith or contribute to the activity or action specified in the disclosurefor the listed elements. Thus, the phrase “consisting essentially of”indicates that the listed elements are required or mandatory, but thatother elements are optional and may or may not be present depending uponwhether or not they affect the activity or action of the listedelements.

The terms “complementary” and “complementarity” refer to polynucleotides(i.e., a sequence of nucleotides) related by the base-pairing rules. Forexample, the sequence “A-G-T,” is complementary to the sequence “T-C-A.”Complementarity may be “partial,” in which only some of the nucleicacids' bases are matched according to the base pairing rules. Or, theremay be “complete” or “total” complementarity between the nucleic acids.The degree of complementarity between nucleic acid strands hassignificant effects on the efficiency and strength of hybridizationbetween nucleic acid strands.

By “corresponds to” or “corresponding to” is meant (a) a polynucleotidehaving a nucleotide sequence that is substantially identical orcomplementary to all or a portion of a reference polynucleotide sequenceor encoding an amino acid sequence identical to an amino acid sequencein a peptide or protein; or (b) a peptide or polypeptide having an aminoacid sequence that is substantially identical to a sequence of aminoacids in a reference peptide or protein.

By “derivative” is meant a polypeptide that has been derived from thebasic sequence by modification, for example by conjugation or complexingwith other chemical moieties (e.g., pegylation) or by post-translationalmodification techniques as would be understood in the art. The term“derivative” also includes within its scope alterations that have beenmade to a parent sequence including additions or deletions that providefor functionally equivalent molecules.

By “enzyme reactive conditions” it is meant that any necessaryconditions are available in an environment (i.e., such factors astemperature, pH, and lack of inhibiting substances) which will permitthe enzyme to function. Enzyme reactive conditions can be either invitro, such as in a test tube, or in vivo, such as within a cell.

As used herein, the terms “function” and “functional” and the like referto a biological, enzymatic, or therapeutic function.

By “gene” is meant a unit of inheritance that occupies a specific locuson a chromosome and consists of transcriptional and/or translationalregulatory sequences and/or a coding region and/or non-translatedsequences (i.e., introns, 5′ and 3′ untranslated sequences).

“Homology” refers to the percentage number of amino acids that areidentical or constitute conservative substitutions. Homology may bedetermined using sequence comparison programs such as GAP (Deveraux etal., 1984, Nucleic Acids Research 12, 387-395) which is incorporatedherein by reference. In this way sequences of a similar or substantiallydifferent length to those cited herein could be compared by insertion ofgaps into the alignment, such gaps being determined, for example, by thecomparison algorithm used by GAP.

The term “host cell” includes an individual cell or cell culture whichcan be or has been a recipient of any recombinant vector(s) or isolatedpolynucleotide of the invention. Host cells include progeny of a singlehost cell, and the progeny may not necessarily be completely identical(in morphology or in total DNA complement) to the original parent celldue to natural, accidental, or deliberate mutation and/or change. A hostcell includes cells transfected or infected in vivo or in vitro with arecombinant vector or a polynucleotide of the invention. A host cellwhich comprises a recombinant vector of the invention is a recombinanthost cell.

By “isolated” is meant material that is substantially or essentiallyfree from components that normally accompany it in its native state. Forexample, an “isolated polynucleotide”, as used herein, refers to apolynucleotide, which has been purified from the sequences which flankit in a naturally-occurring state, e.g., a DNA fragment which has beenremoved from the sequences that are normally adjacent to the fragment.Alternatively, an “isolated peptide” or an “isolated polypeptide” andthe like, as used herein, refer to in vitro isolation and/orpurification of a peptide or polypeptide molecule from its naturalcellular environment, and from association with other components of thecell.

By “increased” or “increasing” is meant the ability of one or moremodified photosynthetic microorganisms, e.g., Cyanobacteria, to producea greater amount of a given fatty acid, lipid molecule, or triglycerideas compared to a control Cyanobacteria, such as an unmodifiedCyanobacteria or a differently modified Cyanobacteria. Production offatty acids can be measured according to techniques known in the art,such as Nile Red staining and gas chromatography. Production oftriglycerides can be measured, for example, using commercially availableenzymatic tests, including colorimetric enzymatic tests usingglycerol-3-phosphate-oxidase.

As used herein, “light harvesting protein” (LHP), means a protein thatmay be part of or associated with a larger supercomplex of aphotosystem, the functional unit in photosynthesis. The LHP is used, forexample, by plants and photosynthetic bacteria to collect more of theincoming light (e.g. photons) than would be captured by thephotosynthetic reaction center alone. LHP may include light harvestingantenna proteins (phycobiliproteins, such as phycocyanin,allophycocyanin, phycoerythrin and evolutionarily relatedphycobiliproteins), enzymes necessary for synthesis of light harvestingchromophores (the bilins, such as phycocyanobilin, phycoerythrobilin,phycourobilin), enzymes necessary for assembling light harvestingchromophores onto phycobiliproteins (known as lyases), light harvestingantenna linker proteins, and chlorophyll binding proteins.

As used herein “light limiting conditions” means that the rate ofphotosynthetic or light harvesting activity and/or carbon fixationassociated with photosynthetic microorganisms is limited by the amountof light available (as opposed to nutrient limiting conditions, or CO2limiting conditions, for example).

As used herein, “neutral lipid” means any lipid that is soluble only insolvents of very low polarity. Neutral lipids are divided into two maingroups: (1) acylglycerols (glycerides), i.e. fatty-acid esters ofglycerol; and (2) waxes, i.e. fatty-acid esters of long-chainmonohydroxy alcohols. More precisely called a fatty acid ester. Arisesfrom the joining of the fatty acid carboxyl group to a hydroxyl group tomake an ester bond. This can occur, for example, between a fatty acidand glycerol to make mono, di and triglycerides, or between a fatty acidand an alcohol to make a wax ester.

By “obtained from” is meant that a sample such as, for example, apolynucleotide extract or polypeptide extract is isolated from, orderived from, a particular source, such as a desired organism or aspecific tissue within a desired organism. “Obtained from” can alsorefer to the situation in which a polynucleotide or polypeptide sequenceis isolated from, or derived from, a particular organism or tissuewithin an organism. For example, a polynucleotide sequence encoding adiacylglycerol acyltransferase, phosphatidate phosphatase, and/oracetyl-CoA carboxylase enzyme may be isolated from a variety ofprokaryotic or eukaryotic organisms, or from particular tissues or cellswithin certain eukaryotic organism.

The term “operably linked” as used herein means placing a gene under theregulatory control of a promoter, which then controls the transcriptionand optionally the translation of the gene. In the construction ofheterologous promoter/structural gene combinations, it is generallypreferred to position the genetic sequence or promoter at a distancefrom the gene transcription start site that is approximately the same asthe distance between that genetic sequence or promoter and the gene itcontrols in its natural setting; i.e. the gene from which the geneticsequence or promoter is derived. As is known in the art, some variationin this distance can be accommodated without loss of function.Similarly, the preferred positioning of a regulatory sequence elementwith respect to a heterologous gene to be placed under its control isdefined by the positioning of the element in its natural setting; i.e.,the genes from which it is derived. “Constitutive promoters” aretypically active, i.e., promote transcription, under most conditions.“Inducible promoters” are typically active only under certainconditions, such as in the presence of a given molecule factor (e.g.,IPTG) or a given environmental condition (e.g., particular CO₂concentration, nutrient levels, light, heat). In the absence of thatcondition, inducible promoters typically do not allow significant ormeasurable levels of transcriptional activity. For example, induciblepromoters may be induced according to temperature, pH, a hormone, ametabolite (e.g., lactose, mannitol, an amino acid), light (e.g.,wavelength specific), osmotic potential (e.g., salt induced), a heavymetal, or an antibiotic. Numerous standard inducible promoters will beknown to one of skill in the art.

The recitation “polynucleotide” or “nucleic acid” as used hereindesignates mRNA, RNA, cRNA, rRNA, cDNA or DNA. The term typically refersto polymeric form of nucleotides of at least 10 bases in length, eitherribonucleotides or deoxynucleotides or a modified form of either type ofnucleotide. The term includes single and double stranded forms of DNA.

The terms “polynucleotide variant” and “variant” and the like refer topolynucleotides displaying substantial sequence identity with areference polynucleotide sequence or polynucleotides that hybridize witha reference sequence under stringent conditions that are definedhereinafter. These terms also encompass polynucleotides that aredistinguished from a reference polynucleotide by the addition, deletionor substitution of at least one nucleotide. Accordingly, the terms“polynucleotide variant” and “variant” include polynucleotides in whichone or more nucleotides have been added or deleted, or replaced withdifferent nucleotides. In this regard, it is well understood in the artthat certain alterations inclusive of mutations, additions, deletionsand substitutions can be made to a reference polynucleotide whereby thealtered polynucleotide retains the biological function or activity ofthe reference polynucleotide, or has increased activity in relation tothe reference polynucleotide (i.e., optimized). Polynucleotide variantsinclude, for example, polynucleotides having at least 50% (and at least51% to at least 99% and all integer percentages in between) sequenceidentity with a reference polynucleotide sequence that encodes adiacylglycerol acyltransferase, a phosphatidate phosphatase, and/or anacetyl-CoA carboxylase enzyme. The terms “polynucleotide variant” and“variant” also include naturally-occurring allelic variants andorthologs that encode these enzymes.

With regard to polynucleotides, the term “exogenous” refers to apolynucleotide sequence that does not naturally occur in a wild-typecell or organism, but is typically introduced into the cell by molecularbiological techniques. Examples of exogenous polynucleotides includevectors, plasmids, and/or man-made nucleic acid constructs encoding adesired protein. With regard to polynucleotides, the term “endogenous”or “native” refers to naturally occurring polynucleotide sequences thatmay be found in a given wild-type cell or organism. For example, certaincyanobacterial species do not typically contain a DGAT gene, and,therefore, do not comprise an “endogenous” polynucleotide sequence thatencodes a DGAT polypeptide. Also, a particular polynucleotide sequencethat is isolated from a first organism and transferred to secondorganism by molecular biological techniques is typically considered an“exogenous” polynucleotide with respect to the second organism.

“Polypeptide,” “polypeptide fragment,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues andto variants and synthetic analogues of the same. Thus, these terms applyto amino acid polymers in which one or more amino acid residues aresynthetic non-naturally occurring amino acids, such as a chemicalanalogue of a corresponding naturally occurring amino acid, as well asto naturally-occurring amino acid polymers. In certain aspects,polypeptides may include enzymatic polypeptides, or “enzymes,” whichtypically catalyze (i.e., increase the rate of) various chemicalreactions.

The recitation polypeptide “variant” refers to polypeptides that aredistinguished from a reference polypeptide sequence by the addition,deletion or substitution of at least one amino acid residue. In certainembodiments, a polypeptide variant is distinguished from a referencepolypeptide by one or more substitutions, which may be conservative ornon-conservative. In certain embodiments, the polypeptide variantcomprises conservative substitutions and, in this regard, it is wellunderstood in the art that some amino acids may be changed to otherswith broadly similar properties without changing the nature of theactivity of the polypeptide. Polypeptide variants also encompasspolypeptides in which one or more amino acids have been added ordeleted, or replaced with different amino acid residues.

The present invention contemplates the use in the methods describedherein of variants of full-length enzymes having, photosynthetic orlight harvesting activity, diacylglycerol acyltransferase activity,phosphatidate phosphatase activity, and/or acetyl-CoA carboxylaseactivity, truncated fragments of these full-length polypeptides,variants of truncated fragments, as well as their related biologicallyactive fragments. Typically, biologically active fragments of apolypeptide may participate in an interaction, for example, anintra-molecular or an inter-molecular interaction. An inter-molecularinteraction can be a specific binding interaction or an enzymaticinteraction (e.g., the interaction can be transient and a covalent bondis formed or broken). Biologically active fragments of apolypeptide/enzyme having a light harvesting activity, diacylglycerolacyltransferase activity, a phosphatidate phosphatase activity, and/oracetyl-CoA carboxylase activity include peptides comprising amino acidsequences sufficiently similar to, or derived from, the amino acidsequences of a (putative) full-length reference polypeptide sequence.Typically, biologically active fragments comprise a domain or motif withat least one activity of a nblA polypeptide, an rpaB polypeptide, a pbsBpolypeptide, a pbsC polypeptide, a Phycobiliprotein polypeptide, adiacylglycerol acyltransferase polypeptide, phosphatidate phosphatasepolypeptide, and/or acetyl-coA carboxylase polypeptide, and may includeone or more (and in some cases all) of the various active domains. Abiologically active fragment of nblA, rpaB, pbsB, pbsC,Phycobiliprotein, diacylglycerol acyltransferase, phosphatidatephosphatase, and/or acetyl-CoA carboxylase polypeptide can be apolypeptide fragment which is, for example, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70,80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240,260, 280, 300, 320, 340, 360, 380, 400, 450, 500, 600 or more contiguousamino acids, including all integers in between, of a referencepolypeptide sequence. In certain embodiments, a biologically activefragment comprises a conserved enzymatic sequence, domain, or motif, asdescribed elsewhere herein and known in the art. Suitably, thebiologically-active fragment has no less than about 1%, 10%, 25%, or 50%of an activity of the wild-type polypeptide from which it is derived.

A “reference sequence,” as used herein, refers to a wild-typepolynucleotide or polypeptide sequence from any organism, e.g., whereinthe polynucleotide encodes a polypeptide having an acyl-ACP reductase,as described herein and known in the art. Exemplary polypeptide“reference sequences” are provided herein, including the polynucleotideand polypeptide sequences of an acyl-ACP reductase of Synechococcuselongatus PCC7942 (see SEQ ID NOs:1 and 2 for the polynucleotide andpolypeptide sequences, respectively) and an acyl-ACP reductase ofSynechocystis sp. PCC6803 (SEQ ID NOs:3 and 4 for the polynucleotide andpolypeptide sequences, respectively), among others known to a personskilled in the art.

The recitations “sequence Identity” or, for example, comprising a“sequence 50% identical to,” as used herein, refer to the extent thatsequences are identical on a nucleotide-by-nucleotide basis or an aminoacid-by-amino acid basis over a window of comparison. Thus, a“percentage of sequence identity” may be calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser,Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn,Gin, Cys and Met) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity.

Terms used to describe sequence relationships between two or morepolynucleotides or polypeptides include “reference sequence”,“comparison window”, “sequence identity”, “percentage of sequenceidentity” and “substantial identity”. A “reference sequence” is at least12 but frequently 15 to 18 and often at least 25 monomer units,inclusive of nucleotides and amino acid residues, in length. Because twopolynucleotides may each comprise (1) a sequence (i.e., only a portionof the complete polynucleotide sequence) that is similar between the twopolynucleotides, and (2) a sequence that is divergent between the twopolynucleotides, sequence comparisons between two (or more)polynucleotides are typically performed by comparing sequences of thetwo polynucleotides over a “comparison window” to identify and comparelocal regions of sequence similarity. A “comparison window” refers to aconceptual segment of at least 6 contiguous positions, usually about 50to about 100, more usually about 100 to about 150 In which a sequence iscompared to a reference sequence of the same number of contiguouspositions after the two sequences are optimally aligned. The comparisonwindow may comprise additions or deletions (i.e., gaps) of about 20% orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by computerized implementations of algorithms (GAP, BESTFIT,FASTA, and TFASTA in the Wisconsin Genetics Software Package Release7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) orby inspection and the best alignment (i.e., resulting in the highestpercentage homology over the comparison window) generated by any of thevarious methods selected. Reference also may be made to the BLAST familyof programs as for example disclosed by Altschul et al., 1997, Nucl.Acids Res. 25:3389. A detailed discussion of sequence analysis can befound in Unit 19.3 of Ausubel et al., “Current Protocols in MolecularBiology”, John Wiley & Sons Inc, 1994-1998, Chapter 15.

As used herein, the term “triglyceride” (triacylglycerol or neutral fat)refers to a fatty acid triester of glycerol. Triglycerides are typicallynon-polar and water-insoluble. Phosphoglycerides (orglycerophospholipids) are major lipid components of biologicalmembranes.

“Transformation” refers to the permanent, heritable alteration in a cellresulting from the uptake and incorporation of foreign DNA into thehost-cell genome; also, the transfer of an exogenous gene from oneorganism into the genome of another organism.

By “vector” is meant a polynucleotide molecule, preferably a DNAmolecule derived, for example, from a plasmid, bacteriophage, yeast orvirus, into which a polynucleotide can be inserted or cloned. A vectorpreferably contains one or more unique restriction sites and can becapable of autonomous replication in a defined host cell including atarget cell or tissue or a progenitor cell or tissue thereof, or beintegrable with the genome of the defined host such that the clonedsequence is reproducible. Accordingly, the vector can be an autonomouslyreplicating vector, i.e., a vector that exists as an extra-chromosomalentity, the replication of which is independent of chromosomalreplication, e.g., a linear or closed circular plasmid, anextra-chromosomal element, a mini-chromosome, or an artificialchromosome. The vector can contain any means for assuringself-replication. Alternatively, the vector can be one which, whenintroduced into the host cell, is integrated into the genome andreplicated together with the chromosome(s) into which it has beenintegrated. Such a vector may comprise specific sequences that allowrecombination into a particular, desired site of the host chromosome. Avector system can comprise a single vector or plasmid, two or morevectors or plasmids, which together contain the total DNA to beintroduced into the genome of the host cell, or a transposon. The choiceof the vector will typically depend on the compatibility of the vectorwith the host cell into which the vector is to be introduced. In thepresent case, the vector is preferably one which is operably functionalin a bacterial cell, such as a cyanobacterial cell. The vector caninclude a reporter gene, such as a green fluorescent protein (GFP),which can be either fused in frame to one or more of the encodedpolypeptides, or expressed separately. The vector can also include aselection marker such as an antibiotic resistance gene that can be usedfor selection of suitable transformants.

The terms “wild-type” and “naturally occurring” are used interchangeablyto refer to a gene or gene product that has the characteristics of thatgene or gene product when isolated from a naturally occurring source. Awild type gene or gene product (e.g., a polypeptide) is that which ismost frequently observed in a population and is thus arbitrarilydesigned the “normal” or “wild-type” form of the gene.

Modified Photosynthetic Microorganisms and Method of Generation Thereof

The present disclosure, therefore, relates generally to modifiedphotosynthetic organisms, including modified Cyanobacteria, and methodsof generation thereof, which have been modified to produce or storereduced levels of light harvesting protein (LHP) as compared towild-type photosynthetic microorganisms. In particular embodiments, themodified photosynthetic organism is genetically modified, for instance,relative to the wild-type or most frequently observed photosyntheticorganism of that same species. Genetic modifications can be man-madeand/or naturally-occurring, for instance, by direct molecular biologicalintervention (e.g., cloning or insertion of exogenous genetic elementsto modulate expression of genes associated with LHP synthesis/storage),directed evolution under controlled conditions to enhance naturalselection of LHP-deficient or LHP-reduced mutants, or identification ofspontaneous LHP-deficient or LHP-reduced mutants under naturalconditions, including combinations thereof. For instance, Cyanobacteria,such as Synechococcus, which contain modulations of the nblA, rpaB,pbsB, pbsC, or Phycobiliprotein gene, individually or in variouscombinations, may produce and accumulate significantly reduced levels ofLHP as compared to wild-type Cyanobacteria. These modifiedCyanoabacterium while producing or storing reduced levels of LHPunexpectedly show increased photosynthetic activity.

Embodiments of the present disclosure include a cell culture comprisingmodified Cyanobacteria that have an increased level of photosyntheticactivity as compared to corresponding wild-type Cyanobacteria, whereinthe modified Cyanobacteria have a reduced amount of a LHP polypeptide ascompared to a corresponding wild-type Cyanobacteria. In particularembodiments, the modified cyanobacteria grow and/or divide at anincreased rate under a condition, such as a light-limited condition, ascompared to the corresponding wild-type cyanobacteria. In particularembodiments, wherein the modified Cyanobacteria have reduced expressionof one or more genes of light harvesting proteins biosynthesis and/ortransportation pathway as compared to the corresponding wild-typeCyanobacteria.

In some embodiments, the modified Cyanobacterium has a reduced level ofexpression of one or more genes of a LHP biosynthesis or storage pathwayand/or overexpresses one or more genes or proteins of a LHP breakdownpathway, such that the modified Cyanobacterium synthesizes oraccumulates a reduced amount of LHP, as compared to a wild-typeCyanobacterium. In one embodiment, the modified Cyanobacterium comprisesone or more mutations or deletions in one or more genes of a LHPbiosynthesis or storage pathway, which may be, e.g., complete or partialgene deletions. In other embodiments, the modified Cyanobacteriacomprise one or more polynucleotides comprising an antisense RNAsequence that targets, e.g., hybridizes to, one or more genes or mRNAsof a LHP biosynthesis or storage pathway, such as an antisenseoligonucleotide or a short interfering RNA (siRNA), or a vector thatexpresses one or more such polynucleotides.

In certain embodiments, individual Cyanobacteria of the modifiedCyanobacteria have a reduced amount of phycobilisomes as compared to thecorresponding wild-type Cyanobacteria. In specific embodiments, themodified Cyanobacteria have an increased proteolytic degradation ofphycobilisomes as compared to the corresponding wild-type Cyanobacteria.In specific embodiments, the modified Cyanobacteria have an increasedexpression of an NbIA gene as compared to the corresponding wild-typeCyanobacteria. In these embodiments, the modified Cyanobacteria have areduction of light harvesting proteins of from 10% to 60%, from 30% to50%, or from 35% to 45%. In certain embodiments, the Cyanobacterium aremodified to have a 40% reduction of light harvesting proteins. In stillother embodiments, the Cyanobacterium is modified to have at least a10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% or 60% reduction oflight harvesting proteins. In yet other embodiments, the Cyanobacteriumis modified to have no more than a 60%, 55%, 50%, 45%, 40%, 35%, 30%,25%, 20%, 15% or 5% reduction of light harvesting proteins. In specificembodiments, the increased expression of the NbIA gene comprises theincreased expression of an endogenous NbIA gene as compared to thecorresponding wild-type Cyanobacteria. In some instances, the expressionof the NbIA gene is increased by replacing a promoter of the NbIA gene.

In certain embodiments, the modified Cyanobacteria have a reduced levelof light harvesting proteins as compared to the corresponding wild-typeCyanobacteria. In specific embodiments, the modified Cyanobacteria havereduced expression of an RpaB gene and/or reduced RpaB activity ascompared to the corresponding wild-type Cyanobacteria. In specificembodiments, the modified Cyanobacteria have over-expressed N terminalfragments of the RpaB gene. In specific embodiments, the level of RpaBactivity is reduced by replacing a promoter of the RpaB gene. Forexample, N terminal fragments of the RpaB gene may be over-expressed byreplacing a promoter of the RpaB gene.

In certain embodiments, the modified Cyanobacteria have a reduced levelof photosystem II light harvesting proteins as compared to thecorresponding wild-type Cyanobacteria. In specific embodiments, themodified Cyanobacteria have a reduced expression of a PbsB gene or aPbsC gene as compared to the corresponding wild-type Cyanobacteria. Inspecific embodiments, the reduced expression of the PbsB gene or thePbsC gene comprises the reduced expression of an endogenous PbsB gene oran endogenous PbsC gene as compared to the corresponding wild-typeCyanobacteria. In some embodiments, the expression of the PbsB gene orthe PbsC gene gene is reduced by replacing a promoter of one of the PbsBgene and the PbsC gene.

In certain embodiments, the modified Cyanobacteria have a reduced amountof phycobiliproteins as compared to the corresponding wild-typeCyanobacteria. In specific embodiments, the modified Cyanobacterium havea reduced expression of phycocyanin genes or allophycocyanin genes ascompared to the corresponding wild-type Cyanobacterium. In specificembodiments, the reduced expression of the phycocyanin genes orallophycocyanin comprises the reduced expression of endogenousphycocyanin genes or allophycocyanin genes as compared to thecorresponding wild-type Cyanobacteria. In some instances, the expressionof the phycocyanin genes or allophycocyanin genes is reduced byreplacing one or more promoters of the phycocyanin genes andallophycocyanin genes.

Embodiments of the present disclosure also include methods forgenerating modified cyanobacteria. In some embodiments, the methodcomprises modifying one or more polynucleotides associated with lightharvesting proteins (LHP) of Cyanobacteria to generate the modifiedCyanobacteria, wherein the modified cyanobacteria have an increasedlevel of photosynthetic activity as compared to corresponding wild-typeCyanobacteria. In these and other embodiments, the method comprisesculturing Cyanobacteria under a stress condition; and isolating modifiedCyanobacteria that have an increased level of photosynthetic activity ascompared to corresponding wild-type Cyanobacteria, wherein the stresscondition comprises culturing under increased light, culturing inmetronidazole containing growth media or both.

In certain embodiments, the culture is maintained at an optical celldensity ranging from 0.25-2.0, 0.5-1.5, or about 1.0, i.e., within 10%of 1.0. In certain embodiments, the cultured modified Cyanobacteria showan increased growth rate, increased oxygen evolution or both whencompared with a corresponding wild-type Cyanobacteria. For example, agrowth rate of the cultured modified Cyanobacteria may be at least 10%or at least 20% greater than a growth rate of a correspondingmicroorganism grown in light limiting conditions. In other embodiments,a growth rate of the microorganism is at least about 1.5, 2, 3, 4, 5, 6,7, 8, 9, 10 or 20-fold greater than a growth rate of a correspondingCyanobacteria.

In certain aspects, the modified photosynthetic organisms describedherein are further modified to increase production of lipids, forinstance, by introducing and/or overexpressing one or more polypeptidesassociated with lipid biosynthesis. Examples of such lipids includefatty acids, fatty alcohols, fatty aldehydes, alkane/alkenes,triglycerides, and wax esters. Hence, in some instances, modifiedphotosynthetic microorganisms that accumulate a reduced amount of LHP ascompared to the wild-type photosynthetic microorganism can furthercomprise one or more introduced or overexpressed polynucleotidesencoding one or more of an acyl carrier protein (ACP), acyl ACP synthase(Aas), acyl-ACP reductase, alcohol dehydrogenase, aldehydedehydrogenase, aldehyde decarbonylase, thioesterase (TES), acetylcoenzyme A carboxylase (ACCase), diacylglycerol acyltransferase (DGAT),phosphatidic acid phosphatase (PAP; or phosphatidate phosphatase),triacylglycerol (TAG) hydrolase, fatty acyl-CoA synthetase,lipase/phospholipase, fatty acyl reductase (FAR) or any combinationthereof.

Certain embodiments thus include modified photosynthetic microorganismsthat accumulate a reduced amount of LHP as compared to the wild-typephotosynthetic microorganism, and which comprise one or more introducedpolynucleotides that encode an enzyme having DGAT activity. Optionally,to further increase production of triglycerides, such photosyntheticmicroorganisms can further comprise one or more introduced oroverexpressed polynucleotides that encode a phosphatidate phosphatase,ACCase, ACP, phospholipase B, phospholipase C, fatty acyl Co-Asynthetase, or any combination thereof. Certain embodiments include anintroduced DGAT in combination with an introduced or overexpressedACCase, PAP, or both.

Certain embodiments of the present disclosure relate to modifiedphotosynthetic organisms, including Cyanobacteria, and methods of usethereof, wherein the modified photosynthetic microorganisms furthercomprise one or more over-expressed, exogenous, or introducedpolynucleotides encoding an acyl-ACP reductase polypeptide, or afragment or variant thereof. In particular embodiments, the fragment orvariant thereof retains at least 50% of one or more activity of thewild-type acyl-ACP reductase polypeptide. As with most any of theoverexpressed polypeptides described herein, an overexpressed acyl-ACPreductase can be encoded by an endogenous or naturally-occurringpolynucleotide which is operably linked to an introduced promoter,typically upstream of the microorganism's natural acyl-ACP reductasecoding region, and/or it can be encoded by an introduced polynucleotidethat encodes an acyl-ACP reductase.

In certain embodiments, an introduced promoter is inducible, and in someembodiments it is constitutive. Included are weak promoters undernon-induced conditions. Exemplary promoters are described elsewhereherein and known in the art. In particular embodiments, the introducedpromoter is exogenous or foreign to the photosynthetic microorganism,i.e., it is derived from a genus/species that differs from themicroorganism being modified. In other embodiments, the introducedpromoter is a recombinantly introduced copy of an otherwise endogenousor naturally-occurring promoter sequence, i.e., it is derived from thesame species of microorganism being modified.

Similar principles can apply to the introduced polynucleotide whichencodes the acyl-ACP reductase or other overexpressed polypeptide (e.g.,aldehyde dehydrogenase). For instance, in particular embodiments, theintroduced polynucleotide encoding the acyl-ACP reductase or otherpolypeptide is exogenous or foreign to the photosynthetic microorganism,i.e., it is derived from a genus/species that differs from themicroorganism being modified. In other embodiments, the introducedpolynucleotide is a recombinantly introduced copy of an otherwiseendogenous or naturally-occurring sequence, i.e., it is derived from thesame species of microorganism being modified.

Acyl-ACP reductase polypeptides, and fragments and variants thereof thatmay be used according to the compositions and methods of the presentdisclosure are described herein. The present disclosure contemplates theuse of naturally-occurring and non-naturally-occurring variants of theseacyl-ACP reductase and other lipid biosynthesis proteins (e.g., ACP,ACCase, DGAT, acyl-CoA synthetase, aldehyde dehydrogenase), as well asvariants of their encoding polynucleotides. These enzyme encodingsequences may be derived from any microorganism (e.g., plants, bacteria)having a suitable sequence, and may also include any man-made variantsthereof, such as any optimized coding sequences (i.e., codon-optimizedpolynucleotides) or optimized polypeptide sequences.

Acyl-ACP reductase polypeptides may also be overexpressed in strains ofphotosynthetic microorganisms that have been modified to overexpress oneor more selected lipid biosynthesis proteins (e.g., selected fatty acidbiosynthesis proteins, triacylglycerol biosynthesis proteins,alkane/alkene biosynthesis proteins, wax ester biosynthesis proteins).

For example, to produce triglycerides, a modified photosyntheticmicroorganism may comprise an overexpressed acyl-ACP reductase incombination with an introduced polynucleotide that encodes a DGAT. Inthese and related embodiments, triglyceride production can be furtherincreased by introduction or overexpression of an aldehydedehydrogenase, for instance, to increase production of fatty acids, theprecursors to triglycerides. One exemplary aldehyde dehydrogenase isencoded by orf0489 of Synechococcus elongatus PCC7942. Also included arehomologs or paralogs thereof, functional equivalents thereof, andfragments or variants thereofs. Functional equivalents can includealdehyde dehydrogenases with the ability to convert acyl aldehydes(e.g., nonyl-aldehyde) into fatty acids. In certain embodiments, thealdehyde dehydrogenase has the amino acid sequence of SEQ ID NO:103(encoded by the polynucleotide sequence of SEQ ID NO:102), or an activefragment or variant of this sequence. These and related embodiments canbe further combined with reduced expression and/or activity of anendogenous aldehyde decarbonylase (e.g., orf1593 in S. elongatus),described herein, to shunt carbon away from alkanes and towards fattyacids, the precursors to triglycerides.

To produce wax esters, a modified photosynthetic microorganism maycomprise an overexpressed acyl-ACP reductase and an introducedpolynucleotide that encodes a DGAT (e.g., a bi-functional DGAT havingwax ester synthase activity) in further combination with an introducedor overexpressed polynucleotide that encodes an alcohol dehydrogenase,such as a long-chain alcohol dehydrogenase. Exemplary alcoholdehydrogenases include slr1192 from Synechocystis sp. PCC6803 andACIAD3612 from Acinetobacter baylii (see SEQ ID NOS:104-107). Alsoincluded are homologs or paralogs thereof, functional equivalentsthereof, and fragments or variants thereofs. Functional equivalents caninclude alcohol dehydrogenases with the ability to convert acylaldehydes (e.g., nonyl-aldehyde, C₁₂, C₁₄, C₁₆, C₁₈, C₂₀ fattyaldehydes) into fatty alcohols, which can then be converted into waxesters by the wax ester synthase. In certain embodiments, the alcoholdehydrogenase has the amino acid sequence of SEQ ID NO:105 (slr1192;encoded by the polynucleotide sequence of SEQ ID NO:104), or an activefragment or variant of this sequence. In some embodiments, the alcoholdehydrogenase has the amino acid sequence of SEQ ID NO:107 (ACIAD3612;encoded by the polynucleotide sequence of SEQ ID NO:106), or an activefragment or variant of this sequence. Certain of these and relatedembodiment can be combined with any one or more of reduced expressionand/or activity of an endogenous aldehyde dehydrogenase (e.g., orf0489deletion) to shunt carbon away from fatty acid production, reducedexpression and/or activity of an endogenous aldehyde decarbonylase(e.g., orf1593 deletion) to shunt carbon away from alkane production, orboth. Also included are combinations that further comprise an introducedor overexpressed acyl carrier protein (ACP), optionally in combinationwith an introduced or overexpressed acyl-ACP synthetase (Aas).

To produce fatty alcohols, a modified photosynthetic microorganism maycomprise an overexpressed acyl-ACP reductase in combination with anintroduced or overexpressed alcohol dehydrogenase. These and relatedembodiments can be further combined with reduced expression and/oractivity of an endogenous aldehyde decarbonylase (e.g., orf1593 from S.elongatus), reduced expression and/or activity of an endogenous aldehydedehydrogenase (e.g., orf489 from S. elongatus), or both, to respectivelyshunt carbon away from alkanes/alkenes and fatty acids and towards fattyalcohols.

To produce alkanes and/or alkenes, a modified photosyntheticmicroorganism may comprise an overexpressed acyl-ACP reductase incombination with an introduced or overexpressed aldehyde decarbonylase.Exemplary aldehyde decarbonylases include that encoded by orf1593 of S.elongatus PCC7942 and its orthologs/paralogs, including those found inSynechocystis sp. PCC6803 (encoded by orfsll0208), N. punctiforme PCC73102, Thermosynechococcus elongatus BP-1, Synechococcus sp. Ja-3-3AB,P. marinus MIT9313, P. marinus NATL2A, and Synechococcus sp. RS 9117,the latter having at least two paralogs (RS 9117-1 and -2). These andrelated embodiments can be further combined with reduced expressionand/or activity of an endogenous aldehyde dehydrogenase (e.g., orf0489from S. elongatus), reduced expression and/or activity of an endogenousalcohol dehydrogenase (e.g., a long-chain alcohol dehydrogenase), orboth, to respectively shunt carbon away from fatty acids and fattyalcohols and towards alkanes and/or alkenes.

To produce fatty acids, such as free fatty acids, a modifiedphotosynthetic microorganism may comprise an overexpressed acyl-ACPreductase in optional combination with an introduced or overexpressedaldehyde dehydrogenase (e.g., orf 0489 from S. elongatus ororthologs/paralogs/homologs thereof). These and related embodiments canbe further combined with reduced expression and/or activity of analdehyde decarbonylase (e.g., orf1593 from S. elongatus), reducedexpression and/or activity of an endogenous alcohol dehydrogenase (e.g.,long-chain alcohol dehydrogenase), or both, to respectively shunt carbonaway from alkanes and fatty alcohols and towards fatty acids. In certainembodiments, such as Cyanobacteria including S. elongatus PCC7942,orf1593 resides directly upstream of orf1594 (acyl-ACP reductase codingregion) and encodes an aldehyde decarbonylase. According to onenon-limiting theory, because the aldehyde decarbonylase encoded byorf1593 utilizes acyl aldehyde as a substrate for alkane production,reducing expression of this protein may further increase yields of freefatty acids by shunting acyl aldehydes (e.g., produced by acyl-ACPreductase) away from an alkane-producing pathway, and towards a fattyacid- or fatty alcohol-producing and storage pathway. PCC7942_orf1593orthologs can be found, for example, in Synechocystis sp. PCC6803(encoded by orfsll0208), N. punctiforme PCC 73102, Thermosynechococcuselongatus BP-1, Synechococcus sp. Ja-3-3AB, P. marinus MIT9313, P.marinus NATL2A, and Synechococcus sp. RS 9117, the latter having atleast two paralogs (RS 9117-1 and -2). Included are strains havingmutations or full or partial deletions of one or more genes encodingthese and other aldehyde decarbonylases, such as S. elongatus PCC7942having a full or partial deletion of orf1593, and Synechocystis sp.PCC6803 having a full or partial deletion of orfsll0208). For instance,an exemplary modified photosynthetic microorganism could comprise anoverexpressed acyl-ACP reductase, combined with a full or partialdeletion of the glgC gene, the glgA gene, and/or the pgm gene,optionally combined with an overexpressed aldehyde dehydrogenase, andoptionally combined with a full or partial deletion of a gene encodingan aldehyde decarbonylase (e.g., PCC7942_orf1593, PCC6803_orfsll0208).

Other combinations include, for example, a modified photosyntheticmicroorganism comprising reduced LHP accumulation, in combination withone more of an overexpressed ACP; an overexpressed acyl-ACP reductase incombination with an overexpressed ACP; an overexpressed acyl-ACPreductase in combination with an overexpressed ACCase; an overexpressedacyl-ACP reductase in combination with an overexpressed ACP and anoverexpressed ACCase; an overexpressed acyl-ACP reductase in combinationwith an overexpressed DGAT and optionally an overexpressed acyl-CoAsynthetase (e.g., a DGAT/acyl-CoA synthetase combination); anoverexpressed acyl-ACP reductase with an overexpressed ACP and anoverexpressed DGAT, optionally combined with an overexpressed acyl-CoAsynthetase; an overexpressed acyl-ACP reductase with an overexpressedACCase and an overexpressed DGAT, optionally in combination with anoverexpressed acyl-CoA synthetase; and an overexpressed acyl-ACPreductase with an overexpressed ACP, overexpressed ACCase, and anoverexpressed DGAT, optionally in combination with an overexpressedacyl-CoA synthetase. Acyl-ACP reductase and DGAT-overexpressing strains,optionally in combination with an overexpressed acyl-CoA synthetase,typically produce increased triglycerides relative to DGAT-onlyoverexpressing strains.

Any one of these embodiments can also be combined with a strain havingreduced expression of an acyl-ACP synthetase (Aas). Without wishing tobe bound by any one theory, an endogenous aldehyde dehydrogenase isacting on the acyl-aldehydes generated by orf1594 and converting them tofree fatty acids. The normal role of such a dehydrogenase might involveremoving or otherwise dealing with damaged lipids. In this scenario, itis then likely that the Aas gene product recycles these free fatty acidsby ligating them to ACP. Accordingly, reducing or eliminating expressionof the Aas gene product might ultimately increase production of fattyacids and thus optionally triglycerides (e.g., in a DGAT-expressingmicroorganism), by reducing or preventing their transfer to ACP.Included are mutations and full or partial deletions of one or more Aasgenes, such as the Aas gene of Synechococcus elongatus PCC 7942. As oneexample, a specific modified photosynthetic microorganism could comprisean overexpressed acyl-ACP reductase, combined with a full or partialdeletion of the glgC gene, the glgA gene, and/or the pgm gene,optionally combined with an overexpressed ACP, ACCase, DGAT/acyl-CoAsynthetase, or all of the foregoing, optionally combined with a full orpartial deletion of a gene encoding an aldehyde decarbonylase (e.g.,PCC7942_orf1593, PCC6803_orfsll0208), and optionally combined with afull or partial deletion of an Aas gene encoding an acyl-ACP synthetase.

Certain embodiments of the systems and methods of the present disclosureutilize modified photosynthetic organisms with reduced LHP accumulationthat are further modified to allow production of isobutanol orisopentanol. In particular embodiments, these organisms comprise one ormore introduced or overexpressed polynucleotides that encode apolypeptide associated with isobutanol or isopentanol production.Examples of such polynucleotides include the genes required to convert a2-keto acid to an aldehyde (2-keto acid decarboxylase) and then convertthe aldehyde to an alcohol (alcohol dehydrogenase) in Synechococcuselongatus, according to Atsumi and Liao 2007 Nature and 2009 NatureBiotech. Expression of these genes, or functional fragments or variantsthereof, should allow for the production of isobutanol or isopentanol(3-methyl-1-butanol). In certain embodiments, these genes areAlpha-ketoisovalerate decarboxylase (2-keto acid decarboxylase) fromLactococcus lactis (kivd) and Alcohol dehydrogenase from E. coli (YqhD).The polynucleotide sequence of Alpha-ketoisovalerate decarboxylase(2-keto acid decarboxylase) from Lactococcus lactis is set forth in SEQID NO:180, and its encoded polypeptide sequence is set forth in SEQ IDNO:181. The polynucleotide sequence of alcohol dehydrogenase from E.coli (YqhD) is set forth in SEQ ID NO:182, and its encoded polypeptidesequence is set forth in SEQ ID NO:183.

In additional related embodiments, the modified photosynthetic organismwith reduced LHP accumulation are further modified to include one ormore introduced or overexpressed polynucleotides involved in convertingpyruvate to the precursors for isobutanol or isopentanol production.Thus, they may also be used in combination with any of the relatedmodifications described above. Examples of such polynucleotides andencoded polypeptides include, acetolactate synthase (e.g., Synechococcuselongatus PCC7942 ilvN (NCBI YP_401451; SEQ ID NO:184)), acetolactatesynthase (e.g., Synechococcus elongatus PCC7942 ilvB (NCBI YP_399158;SEQ ID NO:185)), ketol-acid reductoisomerase (e.g., Synechococcuselongatus PCC7942 ilvC (NCBI YP_400569; SEQ ID NO:186), dihydroxy-aciddehydratase (e.g., Synechococcus elongatus PCC7942 ilvD (NCBI YP_399645;SEQ ID NO:187)), 2-isopropylmalate synthase (e.g., Synechococcuselongatus PCC7942 leuA1 (NCBI YP_399447; SEQ ID NO: 188));2-isopropylmalate synthase (e.g., Synechococcus elongatus PCC7942 leuA2(NCBI YP_400427; SEQ ID NO: 189)), isopropylmalate dehydratase (e.g.,Synechococcus elongatus PCC7942 leuD (NCBIYP_401565; SEQ ID NO:190)),isopropylmalate dehydratase (e.g., Synechococcus elongatus PCC7942 leuC(NCBI YP_400915; SEQ ID NO:191)), 3-isopropylmalate dehydrogenase (e.g.,Synechococcus elongatus PCC7942 leuB (NCBI YP_400522; SEQ ID NO:192);acetolactate synthase (e.g., Bacillus subtilus 168 alsS (NCBI NP_391482;SEQ ID NO:193)); ketol-acid reductoisomerase, NAD(P)-binding (e.g., E.coli K-12, MG1655 ilvC (NCBI NP_418222; SEQ ID NO:194)); anddihydroxyacid dehydratase (e.g., E. coli K-12, MG1655 ilvD (NCBIYP_026248; SEQ DI NO:195)) and functional fragments and variantsthereof.

In additional embodiments, the modified photosynthetic organism withreduced LHP accumulation are further modified to include one or moreintroduced or overexpressed polynucleotides involved in glucosesecretion, in order to allow for continued secretion of glucose from LHPdeficient strains that are placed under stress conditions. Examples ofsuch polynucleotides and encoded polypeptides are glucose permeases andglucose/H+ symporters, such as glcP (e.g., Bacillus subtilis 168 glcP;NCBI NP_388933; SEQ ID NO:176), glcP1 (e.g., Streptomyces coelicolorglcP1; NCBI NP_629713.1; SEQ ID NO:177), glcP2 (e.g., Streptomycescoelicolor A3 glcP2; NCBI NP_631212; SEQ ID NO:178), and Mycobacteriumsmegmatis MC2 155 (NCBI YP_888461; SEQ ID NO:179), and functionalfragments and variants thereof.

Certain embodiments of the systems and methods of the present disclosureutilize modified photosynthetic organisms with reduced LHP accumulationthat are further modified to allow production of 4-hydroxybutyrate. Inparticular embodiments, these photosynthetic organisms comprise one ormore introduced or overexpressed polynucleotides that encode apolypeptide associated with 4-hydroxybutyrate production. Examples ofsuch polynucleotides include the genes required to convert2-oxogluturate into succinate semialdehyde, and then convert the latterinto 4-hydroxybutyrate. In particular embodiments, analpha-ketoglutarate decarboxylase converts 2-oxogluturate into succinatesemialdehyde and a 4-hydroxybutyrate dehydrogenase converts succinatesemialdehyde into 4-hydroxybutyrate. Additional examples of suchpolynucleotides include the genes required to convert succinate intosuccinyl-CoA, convert succinyl-CoA into succinate semialdehyde, and thenconver the latter into 4-hydroxybutyrate. In particular embodiments, asuccinyl-CoA synthetase converts succinate into succinyl-CoA, asuccinate-semialdehyde dehydrogenase converts succinyl-CoA intosuccinate semialdehyde, and a 4-hydroxybutyrate dehydrogenase convertssuccinate semialdehyde into 4-hydroxybutyrate. Specific examples ofalpha-ketoglutarate decarboxylases include those encoded byCCDC5180_0513 (SEQ ID NO:211) from Mycobactertium bovis andSYNPCC7002_A2770 (SEQ ID NO:212) from Synechococcus sp PCC 7002.Specific examples of 4-hydroxybutyrate dehydrogenases include thoseencoded by PGN_0724 (SEQ ID NO:213) from Porphyromonas gingivalis andCKR_2662 (SEQ ID NO:214) from Clostridium kluyveri. Specific examples ofsuccinyl-CoA synthetases include the succinyl-CoA synthetase-alphasubunit encoded by sucC (b0728) (SEQ ID NO:218) from E. coli and thesuccinyl-CoA synthetase-beta subunit encoded by sucD (b0729) (SEQ IDNO:219) from E. coli. Specific examples of succinate-semialdehydedehydrogenases include that encoded by PGTDC60_1813 (SEQ ID NO:220) fromPorphyromonas gingivalis. Expression of certain combinations of these orrelated genes, or functional fragments or variants thereof, should allowfor the production of 4-hydroxybutyrate from 2-oxogluturate orsuccinate.

Certain embodiments of the systems and methods of the present disclosureutilize modified photosynthetic organisms with reduced LHP accumulationthat are further modified to allow production of 4-hydroxybutyrate andoptionally 1,4-butanediol. In some embodiments, and further to thepolypeptides associated with the production of 4-hydroxybutyrate(supra), these microorganisms comprise one or more introduced oroverexpressed polynucleotides that encode a polypeptide associated withthe production of 1,4-butanediol from 4-hydroxybutyrate. Examples ofsuch polynucleotides include the genes required to convert4-hydroxybutyrate into 4-hydroxybutyryl-CoA, then convert4-hydroxybutyryl-CoA into 4-hydroxybutyraldehyde, and then convert4-hydroxybutyraldehyde into 1,4-butanediol. In particular embodiments, a4-hydroxybutyryl-CoA transferase converts 4-hydroxybutyrate into4-hydroxybutyryl-CoA, an aldehyde/alcohol dehydrogenase converts4-hydroxybutyryl-CoA into 4-hydroxybutyraldehyde (e.g., one that iscapable of reducing coA-linked substrates to aldehydes/alcohols), and analdehyde/alcohol dehydrogenase converts 4-hydroxybutyraldehyde into1,4-butanediol. Specific examples of 4-hydroxybutyryl-CoA transferasesinclude that encoded by cat2 (CKR_2666) (SEQ ID NO:215) from Clostridiumkluyveri, including homologs from Clostridium aminobutyricum andPorphyromonas gingivalis. Specific examples of aldehyde/alcoholdehydrogenases include those encoded by adhE2 (CEA_P0034) (SEQ IDNO:216) from Clostridium acetobutylicum and adhE (b1241) (SEQ ID NO:217)from E. coli. Expression of certain combinations of these or relatedgenes, or functional fragments or variants thereof, should allow for theproduction of 4-hydroxybutyrate from 2-oxogluturate or succinate, andthe production of 1,4-butanediol from 4-hydroxybutyrate.

Particular embodiments of the systems and methods of the presentdisclosure utilize modified photosynthetic organisms with reduced LHPaccumulation that are further modified to allow production of polyamineintermediates/precursors. Exemplary polyamine intermediates includeagmatine and putrescine. The systems and methods described herein canproduce increased agmatine and putrescine without any furthermodifications. However, in particular embodiments, to further increaseproduction these microorganisms may comprise one or more introduced oroverexpressed polynucleotides that encode a polypeptide associated withpolyamine intermediate production. Examples of such polynucleotidesinclude the genes required to convert L-arginine into agmatine, andoptionally the genes required to convert agmatine intoN-carbamoylputrescine, and then convert N-carbamoylputrescine intoputrescine. In some embodiments, an arginine decarboxylase is introducedor overexpressed to convert L-arginine into agmatine. In particularembodiments, an agmatine deiminase is introduced or overexpressed toconvert agmatine into N-carbamoylputrescine, and/or aN-carbamoylputrescine amidase is introduced or overexpressed to convertN-carbamoylputrescine into putrescine. Specific examples of argininedecarboxylases include that encoded by Synpcc7942_1037 (SEQ ID NO:221)from S. elongatus PCC7942. Specific examples of agmatine deiminasesinclude that encoded by Synpcc7942_2402 (SEQ ID NO:222) andSynpcc7942_2461 from S. elongatus PCC7942. Specific examples ofN-carbamoylputrescine amidases include that encoded by Synpcc7942_2145(SEQ ID NO:223) from S. elongatus PCC7942. Introduction oroverexpression of certain combinations of these or related genes, orfunctional fragments or variants thereof, should allow for the increasedproduction of agmatine, putrescine, or both.

Increased expression can be achieved a variety of ways, for example, byintroducing a polynucleotide into the photosynthetic organism, modifyingan endogenous gene to overexpress the polypeptide, or both. Forinstance, one or more copies of an otherwise endogenous polynucleotidesequence can be introduced by recombinant techniques to increaseexpression, and/or a promoter/enhancer sequence can be introducedupstream of an endogenous gene to regulate expression.

Modified photosynthetic organisms of the present disclosure may beproduced, for example, using any type of photosynthetic microorganism.These include, but are not limited to photosynthetic bacteria, greenalgae, and Cyanobacteria. The photosynthetic microorganism can be, forexample, a naturally photosynthetic microorganism, such as aCyanobacterium, or an engineered photosynthetic microorganism, such asan artificially photosynthetic bacterium. Exemplary microorganisms thatare either naturally photosynthetic or can be engineered to bephotosynthetic include, but are not limited to, bacteria; fungi;archaea; protists; eukaryotes, such as a green algae; and animals suchas plankton, planarian, and amoeba. Examples of naturally occurringphotosynthetic microorganisms include, but are not limited to, Spirulinamaximum, Spirulina platensis, Dunaliella salina, Botrycoccus braunii,Chlorella vulgaris, Chlorella pyrenoidosa, Serenastrum capricomutum,Scenedesmus auadricauda, Porphyridium cruentum, Scenedesmus acutus,Dunaliella sp., Scenedesmus obliquus, Anabaenopsis, Aulosira,Cylindrospermum, Synechococcus sp., Synechocystis sp., and/orTolypothrix.

A modified Cyanobacteria of the present disclosure may be from anygenera or species of Cyanobacteria that is genetically manipulable,i.e., permissible to the introduction and expression of exogenousgenetic material. Examples of Cyanobacteria that can be engineeredaccording to the methods of the present disclosure include, but are notlimited to, the genus Synechocystis, Synechococcus, Thermosynechococcus,Nostoc, Prochlorococcus, Microcystis, Anabaena, Spirulina, andGloeobacter.

Cyanobacteria, also known as blue-green algae, blue-green bacteria, orCyanophyta, is a phylum of bacteria that obtain their energy throughphotosynthesis. Cyanobacteria can produce metabolites, such ascarbohydrates, proteins, lipids and nucleic acids, from CO₂, water,inorganic salts and light. Any Cyanobacteria may be used according tothe present disclosure.

Cyanobacteria include both unicellular and colonial species. Coloniesmay form filaments, sheets or even hollow balls. Some filamentouscolonies show the ability to differentiate into several different celltypes, such as vegetative cells, the normal, photosynthetic cells thatare formed under favorable growing conditions; akinetes, theclimate-resistant spores that may form when environmental conditionsbecome harsh; and thick-walled heterocysts, which contain the enzymenitrogenase, vital for nitrogen fixation.

Heterocysts may also form under the appropriate environmental conditions(e.g., anoxic) whenever nitrogen is necessary. Heterocyst-formingspecies are specialized for nitrogen fixation and are able to fixnitrogen gas, which cannot be used by plants, into ammonia (NH₃),nitrites (NO₂ ⁻), or nitrates (NO₃ ⁻), which can be absorbed by plantsand converted to protein and nucleic acids.

Many Cyanobacteria also form motile filaments, called hormogonia, whichtravel away from the main biomass to bud and form new colonieselsewhere. The cells in a hormogonium are often thinner than in thevegetative state, and the cells on either end of the motile chain may betapered. In order to break away from the parent colony, a hormogoniumoften must tear apart a weaker cell in a filament, called a necridium.

Each individual Cyanobacterial cell typically has a thick, gelatinouscell wall. Cyanobacteria differ from other gram-negative bacteria inthat the quorum sensing molecules autoinducer-2 and acyl-homoserinelactones are absent. They lack flagella, but hormogonia and someunicellular species may move about by gliding along surfaces. In watercolumns, some Cyanobacteria float by forming gas vesicles, like inarchaea.

Cyanobacteria have an elaborate and highly organized system of internalmembranes that function in photosynthesis. Photosynthesis inCyanobacteria generally uses water as an electron donor and producesoxygen as a by-product, though some Cyanobacteria may also use hydrogensulfide, similar to other photosynthetic bacteria. Carbon dioxide isreduced to form carbohydrates via the Calvin cycle. In most forms, thephotosynthetic machinery is embedded into folds of the cell membrane,called thylakoids. Due to their ability to fix nitrogen in aerobicconditions, Cyanobacteria are often found as symbionts with a number ofother groups of microorganisms such as fungi (e.g., lichens), corals,pteridophytes (e.g., Azolla), and angiosperms (e.g., Gunnera), amongothers.

Cyanobacteria are the only group of microorganisms that are able toreduce nitrogen and carbon in aerobic conditions. The water-oxidizingphotosynthesis is accomplished by coupling the activity of photosystem(PS) II and I (Z-scheme). In anaerobic conditions, Cyanobacteria arealso able to use only PS I (i.e., cyclic photophosphorylation) withelectron donors other than water (e.g., hydrogen sulfide, thiosulphate,or molecular hydrogen), similar to purple photosynthetic bacteria.Furthermore, Cyanobacteria share an archaeal property; the ability toreduce elemental sulfur by anaerobic respiration in the dark. TheCyanobacterial photosynthetic electron transport system shares the samecompartment as the components of respiratory electron transport.Typically, the plasma membrane contains only components of therespiratory chain, while the thylakoid membrane hosts both respiratoryand photosynthetic electron transport.

Phycobilisomes, attached to the thylakoid membrane, act as lightharvesting proteins (e.g. antennae) for the photosystems ofCyanobacteria. The phycobilisome components (phycobiliproteins) areresponsible for the blue-green pigmentation of most Cyanobacteria. Colorvariations are mainly due to carotenoids and phycoerythrins, which mayprovide the cells with a red-brownish coloration. In some Cyanobacteria,the color of light influences the composition of phycobilisomes. Ingreen light, the cells accumulate more phycoerythrin, whereas in redlight they produce more phycocyanin. Thus, the bacteria appear green inred light and red in green light. This process is known as complementarychromatic adaptation and represents a way for the cells to maximize theuse of available light for photosynthesis.

In particular embodiments, the Cyanobacteria may be, e.g., a marine formof Cyanobacteria or a freshwater form of Cyanobacteria. Examples ofmarine forms of Cyanobacteria include, but are not limited toSynechococcus WH8102, Synechococcus RCC307, Synechococcus NKBG 15041c,and Trichodesmium. Examples of freshwater forms of Cyanobacteriainclude, but are not limited to, S. elongatus PCC7942, SynechocystisPCC6803, Plectonema boryanum, and Anabaena sp. Exogenous geneticmaterial encoding the desired enzymes or polypeptides may be introducedeither transiently, such as in certain self-replicating vectors, orstably, such as by integration (e.g., recombination) into theCyanobacterium's native genome.

In other embodiments, a genetically modified Cyanobacteria of thepresent disclosure may be capable of growing in brackish or salt water.When using a freshwater form of Cyanobacteria, the overall net cost forproduction of triglycerides will depend on both the nutrients requiredto grow the culture and the price for freshwater. One can foreseefreshwater being a limited resource in the future, and in that case itwould be more cost effective to find an alternative to freshwater. Twosuch alternatives include: (1) the use of waste water from treatmentplants; and (2) the use of salt or brackish water.

Salt water in the oceans can range in salinity between 3.1% and 3.8%,the average being 3.5%, and this is mostly, but not entirely, made up ofsodium chloride (NaCl) ions. Brackish water, on the other hand, has moresalinity than freshwater, but not as much as seawater. Brackish watercontains between 0.5% and 3% salinity, and thus includes a large rangeof salinity regimes and is therefore not precisely defined. Waste wateris any water that has undergone human influence. It consists of liquidwaste released from domestic and commercial properties, industry, and/oragriculture and can encompass a wild range of possible contaminants atvarying concentrations.

There is a broad distribution of Cyanobacteria in the oceans, withSynechococcus filling just one niche. Specifically, Synechococcus sp.PCC 7002 (formerly known as Agmenellum quadruplicatum strain PR-6) growsin brackish water, is unicellular and has an optimal growing temperatureof 38′C. While this strain is well suited to grow in conditions of highsalt, it will grow slowly in freshwater. In particular embodiments, thepresent disclosure contemplates the use of a Cyanobacteria S. elongatusPCC7942, altered in a way that allows for growth in either waste wateror salt/brackish water. A S. elongatus PCC7942 mutant resistant tosodium chloride stress has been described (Bagchi, S. N. et al.,Photosynth Res. 2007, 92:87-101), and a genetically modified S.elongatus PCC7942 tolerant of growth in salt water has been described(Waditee, R. et al., PNAS 2002, 99:4109-4114). According to the presentdisclosure, a salt water tolerant strain is capable of growing in wateror media having a salinity in the range of 0.5% to 4.0% salinity,although it is not necessarily capable of growing in all salinitiesencompassed by this range. In one embodiment, a salt tolerant strain iscapable of growth in water or media having a salinity in the range of1.0% to 2.0% salinity. In another embodiment, a salt water tolerantstrain is capable of growth in water or media having a salinity in therange of 2.0% to 3.0% salinity.

Examples of Cyanobacteria that may be utilized and/or geneticallymodified according to the methods described herein include, but are notlimited to, Chroococcales Cyanobacteria from the genera Aphanocapsa,Aphanothece, Chamaesiphon, Chroococcus, Chroogloeocystis,Coelosphaerium, Crocosphaera, Cyanobacterium, Cyanobium, Cyanodictyon,Cyanosarcina, Cyanothece, Dactylococcopsis, Gloecapsa, Gloeothece,Merismopedia, Microcystis, Radiocystis, Rhabdoderma, Snowella,Synychococcus, Synechocystis, Thermosenechococcus, and Woronichinia;Nostacales Cyanobacteria from the genera Anabaena, Anabaenopsis,Aphanizomenon, Aulosira, Colothrix, Coleodesmium, Cyanospira,Cylindrospermosis, Cylindrospermum, Fremyella, Gleotrichia, Microchaete,Nodularia, Nostoc, Rexia, Richelia, Scytonema, Sprirestis, andToypothrix; Oscillatoriales Cyanobacteria from the genera Arthrospira,Geitlerinema, Halomicronema, Halospirulina, Katagnymene, Leptolyngbya,Limnothrix, Lyngbya, Microcoleus, Oscillatoria, Phormidium,Planktothricoides, Planktothrix, Plectonema, Pseudoanaboena/Limnothrix,Schizothrix, Spirulina, Symploca, Trichodesmium, Tychonema;Pleurocapsoles cyanobacterium from the genera Chroococcidiopsis,Dermocarpa, Dermocarpella, Myxosarcina, Pleurocapsa, Stanieria,Xenococcus; Prochlorophytes Cyanobacterium from the genera Prochloron,Prochlorococcus, Prochlorothrix; and Stigonematales cyanobacterium fromthe genera Capsosira, Chlorogeoepsis, Fischerella, Hapolosiphon,Mastigocladopsis, Nostochopsis, Stigonema, Symphyonema, Symphonemopsis,Umezakia, and Westiellopsis. In certain embodiments, the Cyanobacteriumis from the genus Synechococcus, including, but not limited toSynechococcus bigranulatus, Synechococcus elongatus, Synechococcusleopoliensis, Synechococcus lividus, Synechococcus nidulans, andSynechococcus rubescens.

In certain embodiments, the Cyanobacterium Is Anabaena sp. strain PCC7120, Synechocystis sp. strain PCC6803, Nostoc muscorum, Nostocellipsosporum, or Nostoc sp. strain PCC 7120. In certain preferredembodiments, the Cyanobacterium is S. elongatus sp. strain PCC7942.

Additional examples of Cyanobacteria that may be utilized in the methodsprovided herein include, but are not limited to, Synechococcus sp.strains WH7803, WH8102, WH8103 (typically genetically modified byconjugation), Baeocyte-forming Chroococcidiopsis spp. (typicallymodified by conjugation/electroporation), non-heterocyst-formingfilamentous strains Planktothrix sp., Plectonema boryanum M101(typically modified by electroporation), and Heterocyst-forming strainsAnabaena sp. strains ATCC 29413 (typically modified by conjugation),Tolypothrix sp. strain PCC 7601 (typically modified byconjugation/electroporation) and Nostoc punctiforme strain ATCC 29133(typically modified by conjugation/electroporation).

In certain preferred embodiments, the Cyanobacterium may be S. elongatussp. strain PCC7942 or Synechococcus sp. PCC 7002 (originally known asAgmenellum quadruplicatum).

In particular embodiments, the genetically modified, photosyntheticmicroorganism, e.g., Cyanobacteria, of the present disclosure may beused to produce triglycerides and/or other carbon-containing compoundsfrom just sunlight, water, air, and minimal nutrients, using routineculture techniques of any reasonably desired scale. In particularembodiments, the present disclosure contemplates using spontaneousmutants of photosynthetic microorganisms that demonstrate a growthadvantage under a defined growth condition. Among other benefits, theability to produce large amounts of triglycerides from minimal energyand nutrient input makes the modified photosynthetic microorganism,e.g., Cyanobacteria, of the present disclosure a readily manageable andefficient source of feedstock in the subsequent production of biofuels,such as biodiesel, and other specialty chemicals, such as glycerin.

Methods of producing a modified photosynthetic microorganism, e.g., aCyanobacterium, that has a reduced light harvesting protein productionas compared to a wild-type photosynthetic microorganism, which may beused in the systems or methods of the present disclosure, includemodifying the photosynthetic microorganism so that it has a reducedlevel of expression of one or more genes of the light harvesting proteinproduction. In certain embodiments, the one or more genes include nblA,rpaB, pbsB, pbsC, Phycobiliprotein gene or a combination thereof. Inparticular embodiments, expression or activity is reduced by mutating ordeleting a portion or all of the one or more genes. In particularembodiments, expression or activity is reduced by knocking out orknocking down one or more alleles of the one or more genes. Inparticular embodiments, expression or activity of the one or more genesis reduced by contacting the photosynthetic microorganism with anantisense oligonucleotide or interfering RNA, e.g., an siRNA, thattargets the one or more genes. In particular embodiments, a vector thatexpresses a polynucleotide that hybridizes to the one or more genes,e.g., an antisense oligonucleotide or an siRNA is introduced into thephotosynthetic microorganism.

In certain embodiments, the method comprises modifying one or morepolynucleotides associated with a light harvesting protein (LHP) ofCyanobacteria to generate the modified Cyanobacteria, wherein themodified cyanobacteria have an increased level of photosyntheticactivity as compared to corresponding wild-type Cyanobacteria. In theseembodiments and other embodiments, the method comprises culturingCyanobacteria under a stress condition; and isolating modifiedCyanobacteria that have an increased level of photosynthetic activity ascompared to corresponding wild-type Cyanobacteria, wherein the stresscondition comprises culturing under increased light, culturing inmetronidazole containing growth media or both.

In some embodiments, the photosynthetic activity of the Cyanobacteria isgreater than photosynthetic activity of the corresponding wild-typeCyanobacteria. In certain embodiments, the photosynthetic activity ismeasured based on at least one of a growth rate, a level of oxygenevolution, or a biomass accumulation rate. In particular embodiments,the growth rate of the modified Cyanobacteria is at least about 110% ofa growth rate of the corresponding wild-type Cyanobacteria. Inparticular embodiments, the growth rate of the modified Cyanobacteria isat least about 120% of a growth rate of the corresponding wild-typeCyanobacteria. In particular embodiments, the growth rate of themodified Cyanobacteria is at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10or 20-fold greater than a growth rate of the corresponding wild-typeCyanobacteria. In particular embodiments, the growth rate is measured atabout day 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 post-initiationof culturing.

In certain embodiments, the level of oxygen evolution of the modifiedCyanobacteria is at least about 110% of a level of oxygen evolution ofthe corresponding wild-type Cyanobacteria. In particular embodiments,the level of oxygen evolution of the modified Cyanobacteria is at leastabout 120% of a level of oxygen evolution of the corresponding wild-typeCyanobacteria. In particular embodiments, the level of oxygen evolutionof the modified Cyanobacteria is at least about 1.5, 2, 3, 4, 5, 6, 7,8, 9, 10 or 20-fold greater than a level of oxygen evolution of thecorresponding wild-type Cyanobacteria. In particular embodiments, thelevel of oxygen evolution is measured at about day 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, or 14 post-initiation of culturing.

In certain embodiments, the biomass accumulation rate of the modifiedCyanobacteria is at least about 110% of a biomass accumulation rate ofthe corresponding wild-type Cyanobacteria. In particular embodiments,the biomass accumulation rate of the modified Cyanobacteria is at leastabout 120% of a level of biomass accumulation of the correspondingwild-type Cyanobacteria. In particular embodiments, the biomassaccumulation rate of the modified Cyanobacteria is at least about 1.5,2, 3, 4, 5, 6, 7, 8, 9, 10 or 20-fold greater than a biomassaccumulation rate of the corresponding wild-type Cyanobacteria. Inparticular embodiments, the biomass accumulation rate is measured atabout day 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 post-initiationof culturing.

Photosynthetic microorganisms, e.g., Cyanobacteria may be geneticallymodified according to techniques known in the art, e.g., to delete aportion or all of a gene or to introduce a polynucleotide that expressesa functional polypeptide. As noted above, in certain aspects, geneticmanipulation in photosynthetic microorganisms, e.g., Cyanobacteria, canbe performed by the introduction of non-replicating vectors whichcontain native photosynthetic microorganism sequences, exogenous genesof interest, and selectable markers or drug resistance genes. Uponintroduction into the photosynthetic microorganism, the vectors may beintegrated into the photosynthetic microorganism's genome throughhomologous recombination. In this way, an exogenous gene of interest andthe drug resistance gene are stably integrated into the photosyntheticmicroorganism's genome. Such recombinants cells can then be isolatedfrom non-recombinant cells by drug selection. Cell transformationmethods and selectable markers for Cyanobacteria are also well known inthe art (see, e.g., Wirth, Mol Gen Genet 216:175-7, 1989; andKoksharova, Appl Microbiol Biotechnol 58:123-37, 2002; and THECYANOBACTERIA: MOLECULAR BIOLOGY, GENETICS, AND EVOLUTION (eds. AntonioHerrera and Enrique Flores) Calster Academic Press, 2008, each of whichis incorporated by reference for their description on gene transfer intoCyanobacteria, and other information on Cyanobacteria).

Generation of deletions or mutations of any of the one or more genesassociated with the light harvesting protein production or lipidbiosynthesis can be accomplished according to a variety of methods knownin the art, including those described and exemplified herein. Forinstance, the instant application describes the use of anon-replicating, selectable vector system that is targeted to theupstream and downstream flanking regions of a given gene (e.g., nblA,rpaB), and which recombines with the Cyanobacterial genome at thoseflanking regions to replace the endogenous coding sequence with thevector sequence. Given the presence of a selectable marker in the vectorsequence, such as a drug selectable marker, Cyanobacterial cellscontaining the gene deletion can be readily isolated, identified andcharacterized. Such selectable vector-based recombination methods neednot be limited to targeting upstream and downstream flanking regions,but may also be targeted to internal sequences within a given gene, aslong as that gene is rendered “non-functional,” as described herein.

The generation of deletions or mutations can also be accomplished usingantisense-based technology. For instance, Cyanobacteria have been shownto contain natural regulatory events that rely on antisense regulation,such as a 177-nt ncRNA that is transcribed in antisense to the centralportion of an iron-regulated transcript and blocks its accumulationthrough extensive base pairing (see, e.g., Dühring, et al., Proc. Natl.Acad. Sci. USA 103:7054-7058, 2006), as well as a alr1690 mRNA thatoverlaps with, and is complementary to, the complete furA gene, whichacts as an antisense RNA (α-furA RNA) interfering with furA transcripttranslation (see, e.g., Hernandez et al., Journal of Molecular Biology355:325-334, 2006). Thus, the incorporation of antisense moleculestargeted to genes involved in the light harvesting protein production orlipid biosynthesis would be similarly expected to negatively regulatethe expression of these genes, rendering them “non-functional,” asdescribed herein.

As used herein, antisense molecules encompass both single anddouble-stranded polynucleotides comprising a strand having a sequencethat is complementary to a target coding strand of a gene or mRNA. Thus,antisense molecules include both single-stranded antisenseoligonucleotides and double-stranded siRNA molecules.

In certain aspects, modified photosynthetic microorganisms, e.g.,Cyanobacteria, that may be used in the systems and methods of thepresent disclosure may be prepared by: (i) modifying a photosyntheticmicroorganism so that it expresses a reduced amount of one or more genesassociated with the light harvesting protein production or storagepathway and/or expresses an increased amount of one or morepolynucleotides encoding a polypeptide associated with the lightharvesting protein breakdown pathway or secretion of the lightharvesting protein precursor; and (ii) introducing into thephotosynthetic microorganism one or more polynucleotides encoding one ormore enzymes associated with lipid biosynthesis, secretion of glucose,isobutanol and/or isopentanol biosynthesis, 4-hydroxybutyrate and/or1,4-butanediol biosynthesis, or polyamine intermediate biosynthesis, asdescribed elsewhere herein, and/or (iii) introducing into thephotosynthetic microorganism one or more polynucleotide regulatoryelements (e.g., promoters, enhancers) that increase or otherwiseregulate expression of one or more endogenous enzymes associated withlipid biosynthesis, secretion of glucose, isobutanol and/or isopentanolbiosynthesis, 4-hydroxybutyrate and/or 1,4-butanediol biosynthesis, orpolyamine intermediate biosynthesis; and/or (iv) modifying aphotosynthetic microorganism so that it expresses a reduced amountand/or a reduced-function mutant of one or more selectedgenes/polypeptides associated with lipid biosynthesis, as describedherein. The methods may further comprise a step of: (v) selecting forphotosynthetic microorganisms in which the one or more desiredpolynucleotides were successfully introduced, where the polynucleotideswere, e.g., present in a vector that expressed a selectable marker, suchas an antibiotic resistance gene. As one example, selection andisolation may include the use of antibiotic resistant markers known inthe art (e.g., kanamycin, spectinomycin, and streptomycin).

Other modifications described herein may be produced using standardprocedures and reagents, e.g., vectors, available in the art. Relatedmethods are described in PCT Application No. WO 2010/075440, which ishereby incorporated by reference in its entirety.

The photosynthetic microorganisms and methods of the present disclosuremay be used to produce lipids, such as fatty acids, triglycerides,alkanes/alkenes, fatty alcohols, and/or wax esters. Accordingly, thepresent disclosure provides methods of producing lipids comprisingculturing any of the modified photosynthetic microorganisms describedherein wherein the modified photosynthetic microorganism produces,secretes and/or accumulates (e.g., stores,) an increased amount ofcellular lipid as compared to a corresponding wild-type or unmodifiedphotosynthetic microorganism.

In one embodiment, the modified photosynthetic microorganism is aCyanobacterium that produces or accumulates increased fatty acidsrelative to an unmodified or wild-type Cyanobacterium of the samespecies. In certain embodiments, the modified photosyntheticmicroorganism such as Cyanobacteria produces increased levels ofparticular fatty acids, such as C16:0 fatty acids. In certainembodiments, the modified photosynthetic microorganism is aCyanobacterium that produces or accumulates increased wax estersrelative to an unmodified or wild-type Cyanobacterium of the samespecies. In particular embodiments, the modified photosyntheticmicroorganism is a Cyanobacterium that produces or accumulates increasedtriglycerides relative to an unmodified or wild-type Cyanobacterium ofthe same species. In some embodiments, the modified photosyntheticmicroorganism is a Cyanobacterium that produces or accumulates increasedalkanes and/or alkenes relative to an unmodified or wild-typeCyanobacterium of the same species.

In certain embodiments, the one or more introduced polynucleotides arepresent in one or more expression constructs. In particular embodiments,the one or more expression constructs comprises one or more induciblepromoters. In certain embodiments, the one or more expression constructsare stably integrated into the genome of the modified photosyntheticmicroorganism. In certain embodiments, the introduced polynucleotideencoding an introduced protein is present in an expression constructcomprising a weak promoter under non-induced conditions. In certainembodiments, one or more of the introduced polynucleotides arecodon-optimized for expression in a Cyanobacterium, e.g., aSynechococcus elongatus.

In particular embodiments, the photosynthetic microorganism is aSynechococcus elongatus, such as Synechococcus elongatus strain PCC7942or a salt tolerant variant of Synechococcus elongatus strain PCC7942.

In particular embodiments, the photosynthetic microorganism is aSynechococcus sp. PCC 7002 or a Synechocystis sp. PCC6803.

In particular embodiments, the modified photosynthetic microorganismsare cultured under conditions suitable for inducing expression of theintroduced polynucleotide(s), e.g., wherein the introducedpolynucleotide(s) comprise an inducible promoter. Conditions andreagents suitable for inducing inducible promoters are known andavailable in the art. Also included are the use of auto-inductivesystems, for example, where a metabolite represses expression of theintroduced polynucleotide, and the use of that metabolite by themicroorganism over time decreases its concentration and thus itsrepressive activities, thereby allowing increased expression of thepolynucleotide sequence.

In certain embodiments, modified photosynthetic microorganisms, e.g.,Cyanobacteria, are grown under conditions favorable for producinglipids, triglycerides and/or fatty acids. In particular embodiments,light intensity is between 100 and 2000 uE/m2/s, or between 200 and 1000uE/m2/s. In particular embodiments, the pH range of culture media isbetween 7.0 and 10.0. In certain embodiments, CO₂ is injected into theculture apparatus to a level in the range of 1% to 10%. In particularembodiments, the range of CO₂ is between 2.5% and 5%. In certainembodiments, nutrient supplementation is performed during the linearphase of growth. Each of these conditions may be desirable fortriglyceride production.

In certain embodiments, the modified photosynthetic microorganisms arecultured, at least for some time, under static growth conditions asopposed to shaking conditions. For example, the modified photosyntheticmicroorganisms may be cultured under static conditions prior to inducingexpression of an introduced polynucleotide (e.g., acyl-ACP reductase,ACP, LHP breakdown protein, ACCase, DGAT, fatty acyl-CoA synthetase,aldehyde dehydrogenase, alcohol dehydrogenase, aldehyde decarbonylase)and/or the modified photosynthetic microorganism may be cultured understatic conditions while expression of an introduced polynucleotide isbeing induced, or during a portion of the time period during whichexpression of an introduced polynucleotide is being induced. Staticgrowth conditions may be defined, for example, as growth without shakingor growth wherein the cells are shaken at less than or equal to 30 rpmor less than or equal to 50 rpm.

In certain embodiments, the modified photosynthetic microorganisms arecultured, at least for some time, in media supplemented with varyingamounts of bicarbonate. For example, the modified photosyntheticmicroorganisms may be cultured with bicarbonate at 5, 10, 20, 50, 75,100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 mM bicarbonate priorto inducing expression of an introduced polynucleotide (e.g., acyl-ACPreductase, ACP, LHP breakdown protein, ACCase, DGAT, fatty acyl-CoAsynthetase, alcohol dehydrogenase, aldehyde dehydrogenase, aldehydedecarbonylase) and/or the modified photosynthetic microorganism may becultured with aforementioned bicarbonate concentrations while expressionof an introduced polynucleotide is being induced, or during a portion ofthe time period during which expression of an introduced polynucleotideis being induced.

In related embodiments, modified photosynthetic microorganisms andmethods of the present disclosure may be used in the production of abiofuel or other specialty chemical. Thus, in particular embodiments, amethod of producing a biofuel comprises culturing any of the modifiedphotosynthetic microorganisms of the present disclosure under conditionswherein the modified photosynthetic microorganism accumulates anincreased amount of total cellular lipid (e.g., fatty acid, wax ester,alkane/alkene, fatty alcohol, and/or triglyceride), as compared to acorresponding wild-type photosynthetic microorganism, obtaining thecellular lipid from the microorganism, and processing the obtainedcellular lipid to produce a biofuel. In another embodiment, a method ofproducing a biofuel comprises processing lipids (e.g., fatty acids, waxesters, alkanes/alkenes, fatty alcohols, triglycerides) produced by amodified photosynthetic microorganism of the present disclosure toproduce a biofuel. In particular embodiments, the modifiedphotosynthetic microorganism is grown under stress conditions wherein ithas reduced growth but maintains photosynthesis.

Methods of processing lipids from microorganisms to produce a biofuel orother specialty chemical, e.g., biodiesel, are known and available inthe art. For example, triglycerides may be transesterified to producebiodiesel. Transesterification may be carried out by any one of themethods known in the art, such as alkali-, acid-, or lipase-catalysis(see, e.g., Singh et al. Recent Pat Biotechnol. 2008, 2(2):130-143).Various methods of transesterification utilize, for example, use of abatch reactor, a supercritical alcohol, an ultrasonic reactor, ormicrowave irradiation (Such methods are described, for example, in Jeongand Park, Appl Biochem Biotechnol. 2006, 131(1-3):668-679; Fukuda etal., Journal of Bioscience and Engineering. 2001, 92(5):405-416; Shahand Gupta, Chemistry Central Journal. 2008, 2(1):1-9; and Carrillo-Munozet al., J Org Chem. 1996, 61(22):7746-7749). The biodiesel may befurther processed or purified, e.g., by distillation, and/or a biodieselstabilizer may be added to the biodiesel, as described, for example, inU.S. Patent Application Publication No. 2008/0282606.

Polypeptides

Embodiments of the present disclosure include modified photosyntheticmicroorganisms, such as Cyanobacteria that have modulated the expressionlevel of certain genes involved in light harvesting proteins (LHP)synthesis, such as by mutation or deletion, leads to reduced LHPsynthesis and/or storage in the modified photosynthetic microorganisms.For instance, Cyanobacteria, such as Synechococcus, which containmodulations of the nblA, rpaB, pbsB, pbsC, or Phycobiliprotein gene,individually or in various combinations, may produce and accumulatesignificantly reduced levels of LHP as compared to wild-typeCyanobacteria.

Further to including a reduced LHP, the modified photosyntheticmicroorganisms include diacylglycerol acyltransferase (DGAT) fusionproteins, comprising at least one DGAT polypeptide fused to at least oneheterologous intracellular localization domain, such as a bacterialmembrane-targeting domain. Such fusion proteins can be partially orfully isolated from other cellular components, or expressed, forexample, in cell-free systems or a host cell, such as a modifiedphotosynthetic microorganism.

In certain instances, the modified photosynthetic microorganismsdescribed herein can optionally comprise any combination of one or moreoverexpressed or introduced lipid biosynthesis proteins and/or one ormore overexpressed or introduced proteins associated with glycogenbreakdown. Examples of lipid biosynthesis proteins include acyl carrierproteins (ACP), acyl ACP synthases (Aas), acyl-ACP reductases, alcoholdehydrogenases, aldehyde dehydrogenases, aldehyde decarbonylases,thioesterases (TES), acetyl coenzyme A carboxylases (ACCase),phosphatidic acid phosphatases (PAP; or phosphatidate phosphatases),triacylglycerol (TAG) hydrolases, fatty acyl-CoA synthetases, andlipases/phospholipases, as described herein. Exemplary proteinsassociated with glycogen breakdown are described infra.

In certain instances, photosynthetic microorganisms may optionallycomprise reduced, eliminated, or non-functional expression (e.g.,expression of a deletion mutant with reduced or no functional activity)of one or more endogenous lipid biosynthesis proteins. In particularaspects, for example, in the production of wax esters, modifiedphotosynthetic microorganisms such as Synechococcus may optionallycomprise reduced, eliminated, or non-functional expression of one ormore aldehyde decarbonylases (e.g., orf1593), aldehyde dehydrogenases(e.g., orf0489), or both. In certain aspects, a modified photosyntheticmicroorganism may optionally comprise reduced, eliminated, ornon-functional expression of an Aas polypeptide.

Any of these modified photosynthetic microorganisms may optionallycomprise reduced, eliminated, or non-functional expression of one ormore proteins associated with glycogen biosynthesis, either alone or incombination with overexpressed lipid biosynthesis proteins and/oroverexpressed glycogen breakdown proteins, or in combination with anyother polypeptide-related modification described herein.

As will be apparent, modified photosynthetic microorganisms of thepresent disclosure may comprise any combination of one or more of theadditional modifications noted herein, typically as long as they expressat least one intracellular localization domain-DGAT fusion protein. Itis further understood that the compositions and methods of the presentdisclosure may be practiced using biologically active variants and/orfragments of any of the polypeptides described herein.

(i) Intracellular Localization Domain-DGAT Fusion Proteins

As noted above, embodiments of the present disclosure includeintracellular localization domain-DGAT “fusion proteins,” comprising atleast one DGAT polypeptide fused to at least one heterologousintracellular localization domain, such as a bacterialmembrane-targeting domain.

“Fusion proteins” are defined elsewhere herein and well known in theart, as are methods of making fusion proteins. Fusion proteins may beprepared using standard techniques. For example, DNA sequences encodingthe polypeptide components of a desired fusion may be assembledseparately, and ligated into an appropriate expression vector. The 3′end of the DNA sequence encoding one polypeptide component can beligated, with or without a peptide linker (described below), to the 5′end of a DNA sequence encoding the second (or third, fourth, etc.)polypeptide component so that the reading frames of the sequences are inphase. This permits translation into a single fusion protein thatretains the biological activity of both component polypeptides.

The ligated DNA sequences may be operably linked to suitabletranscriptional or translational regulatory elements. The regulatoryelements responsible for expression of DNA are typically located 5′ tothe DNA sequence encoding the first polypeptide (e.g., themembrane-targeting domain). Similarly, stop codons required to endtranslation and transcription termination signals are typically present3′ to the DNA sequence encoding the second (or third, fourth, etc.)polypeptide.

In the DGAT fusion proteins described herein, the intracellularlocalization or targeting domain can be fused to the N-terminus of theDGAT polypeptide, the C-terminus of the DGAT polypeptide, internally, orany combination thereof. For internal fusions, the intracellularlocalization or targeting domain can be fused to the DGAT polypeptidewithin the N-terminal region (e.g., within about the first 2, 3, 4, 5,6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or so amino acids),at an internal region (between the N-terminal and C-terminal regions),and/or within the C-terminal region (e.g., within about the last 2, 3,4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or so aminoacids). Preferably, the intracellular localization or targeting domainis fused to the N-terminus of the DGAT polypeptide, or near theN-terminus, for example, within about the 1-100 amino acids. Inparticular embodiments, the intracellular localization or targetingdomain is fused to the second amino acid of a DGAT polypeptide,resulting in the removal of the first residue (the methionine residue ofthe AUG codon).

The intracellular localization domains described herein alter theintracellular localization of the DGAT protein(s) to which they arefused. Such alterations can thus be measured relative to thelocalization of the corresponding wild-type DGAT protein(s). In the mostgeneral aspects, a DGAT fusion protein is “targeted to” or “selectivelylocalizes” to one or more defined intracellular region(s) of aphotosynthetic microorganism, where at least about 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, 98%, or nearly 100% of the DGAT fusion protein canbe found associated with the defined intracellular region(s), relativeto a cytoplasmic or a soluble fraction of the microorganism. Inparticular aspects, the intracellular region is one or more of theplasma membrane, a thylakoid, a vesicle, a lipid body, a glycogengranule, a polyhydroxybutyrate (PHB) body, a carboxysome, a cyanophycingranule, and/or an intracellular membrane, such as an intracellularmembrane associated with a thylakoid, vesicle, lipid body, glycogengranule, PHB body, carboxysome, and/or cyanophycin granule. In certainembodiments, the enzymatic domain(s) of the DGAT fusion proteinselectively localizes to the cytoplasmic side of the intracellularregion(s) or associated membranes.

In more particular embodiments, a DGAT fusion protein is “targeted to”or “selectively localizes” to one or more membrane(s) of aphotosynthetic microorganism, where at least about 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, 98%, or nearly 100% of the fusion protein can befound associated with at least one membrane (e.g., a membrane fraction)upon expression in a given photosynthetic microorganism (e.g.,Cyanobacteria), relative to other cellular spaces, such as a cytoplasmicor soluble fraction of the cell. Examples of membranes include theplasma membrane and any intracellular membranes, such as intracellularmembranes associated with a thylakoid, vesicle, lipid body, glycogengranule, PHB body, carboxysome, and/or cyanophycin granule. In someembodiments, the enzymatic domain(s) of the DGAT fusion proteinselectively localize to the cytoplasmic side of the membrane.

In certain embodiments, a DGAT fusion protein is “targeted to” or“selectively localizes” to the plasma membrane of a photosyntheticmicroorganism, where at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 98%, or nearly 100% of the fusion protein can be found at theplasma membrane (or associated with the plasma membrane) upon expressionin a given photosynthetic microorganism (e.g., Cyanobacteria), relativeto other cellular spaces, such as the cytoplasm, vesicles, lipid bodies,thylakoids, glycogen granules, PHB bodies, carboxysomes, cyanophycingranules, other intracellular membranes (e.g., thylakoid membranes,lipid body membranes, vesicle membranes), or any combination thereof.Thylakoids consist of a thylakoid membrane surrounding a thylakoidlumen, and are the site of photosynthesis. In certain of these andrelated embodiments, the enzymatic domain(s) of the DGAT fusion proteinselectively localize to the cytoplasmic side of the plasma membrane.

In particular aspects, the fusion to a heterologous intracellularlocalization domain limits the potential for DGAT-mediated photosystemdisruption, generation of reactive oxygen species, and loss of cellviability, and thereby improves the cell growth phenotype ofDGAT-expressing and lipid-producing photosynthetic microorganisms.

Intracellular Localization Domains.

Generally, the intracellular localization domain sequences of the DGATfusion proteins described herein can be obtained from any one or moresignal or other sequences that selectively localize a given protein to adefined intracellular region (for instance, relative to dispersalthroughout the cytoplasm), such as the plasma membrane, a thylakoid, avesicle, a lipid body, a glycogen granule, a PHB body, a carboxysome, acyanophycin granule, or an intracellular membrane. Particular examplesthus include membrane-, thylakoid-, vesicle-, lipid body-, glycogengranule-, polyhydroxybutyrate (PHB) body-, carboxysome-, and cyanophycingranule-targeting domains, including domains that target DGAT to themembranes associated with these intracellular regions. In specificinstances, the intracellular localization domain selectively localizesthe active domain(s) of DGAT to the cytoplasmic side of theintracellular region, so that DGAT can interact with lipid-producingsubstrates in the cytoplasm.

The intracellular localization domain can be any length that issufficient to selectively localize the DGAT fusion protein(s) to anintracellular region of a membrane, such as the plasma membrane, and/oralter its relation to other cell membranes or substrates, and allow theenzymatic portions of the DGAT polypeptide to interact withlipid-producing substrates in the cytoplasm. For instance, in certainembodiments, the intracellular localization domain can be anywhere fromabout 10-1000 amino acids in length, including about 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130,140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270,280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410,420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550,560, 570, 580, 590, 600, 700, 710, 720, 730, 740, 750, 760, 770, 780,790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920,930, 940, 950, 960, 970, 980, 990, 1000 or more amino acids in length,including all integers and ranges in between (e.g., 20-100, 30-100,40-100, 50-100, 20-200, 30-200, 40-200, 50-200 amino acids in length).

In particular embodiments, the intracellular localization domain is amembrane-targeting or plasma membrane (PM)-targeting domain. Suchmembrane-targeting sequences can be obtained or derived from anycombination of N-terminal leader sequence(s), transmembrane domainsequence(s), and/or integral membrane sequence(s) of a bacterialmembrane protein, such as a bacterial plasma membrane protein. Incertain instances, such bacterial plasma membrane proteins (in theirendogenous state) selectively localize to the plasma membrane, and arecharacterized by having at least one C-terminal region that is localizedto the cytoplasmic side of a bacterial plasma membrane, and/or theperiplasmic side of the outer membrane (for plasma membrane proteinsderived from gram-negative bacteria).

A bacterial plasma membrane protein “selectively localizes” to theplasma membrane where at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 98%, or nearly 100% of the protein can be found at the plasmamembrane upon (preferably endogenous) expression in the bacteria fromwhich it is derived (e.g., gram-positive bacteria, gram-negativebacteria, photosynthetic bacteria, Cyanobacteria), relative to othercellular spaces, such as the cytoplasm, the cell wall, other cellular‘organelles’ or membranes, such as thylakoid membranes for certainphotosynthetic bacteria, or any combination thereof.

In certain embodiments, the membrane-targeting or PM-targeting domainmay comprise an amino acid sequence of an N-terminal leader sequence, anamino acid sequence of one or more transmembrane domains (e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, 10 or more transmembrane domains), an amino acidsequence of one or more integral membrane domains, or any combinationthereof. When combined, the sequences of the N-terminal leader, thetransmembrane domain(s), and/or the integral membrane domain(s) can befrom the same or different bacterial plasma membrane protein(s).

Membrane-targeting or PM-targeting domain sequences can be obtained from(or derived from) the signal sequences, transmembrane domains, orintegral membrane domains of any variety of bacterial membrane proteins.For instance, the bacterial membrane protein can be an integral membraneprotein (IMP), such as a transmembrane protein (TP). In some instances,the membrane-targeting domain is obtained from a single-passtransmembrane protein, having only one domain that spans the lipidbilayer of the plasma membrane, or a multi-pass transmembrane protein,having about 2, 3, 4, 5, 6, 7, 8, 9, 10 or more domains that span thelipid bilayer of the plasma membrane. For the latter, themembrane-targeting domain can comprise any one or more of the multipletransmembrane domains. In certain aspects, the membrane-targeting domainor transmembrane domain (TMD) can comprise an alpha-helicaltransmembrane structure, or a beta-barrel transmembrane structure, thelatter typically deriving from gram-negative outer membrane proteins.

In some embodiments, the membrane-targeting or PM-targeting domain doesnot span the entire lipid bilayer, but inserts into or attaches to thecytoplasmic side of the membrane, such as the plasma membrane. Examplesinclude membrane-targeting domains that interact with the membrane by anamphipathic helix (e.g., parallel to the membrane plane),membrane-targeting domains that interact with the membrane by ahydrophobic loop, and membrane-targeting domains that interact with themembrane by electrostatic or ionic interactions, for example, throughcalcium ions.

In some embodiments, the membrane-targeting domain sequence is obtainedfrom a membrane protein or plasma membrane protein of one or moregram-negative bacteria, gram-positive bacteria, or other bacteria, suchas a Cyanobacteria. Exemplary bacteria are described elsewhere hereinand known in the art.

In particular embodiments the membrane-targeting or PM-targeting domainsequence is obtained from a membrane protein or plasma membrane proteinof a photosynthetic bacteria, such as a Cyanobacteria from the generaAphanocapsa, Aphanothece, Chamaesiphon, Chroococcus, Chroogloeocystis,Coelosphaerium, Crocosphaera, Cyanobacterium, Cyanobium, Cyanodictyon,Cyanosarcina, Cyanothece, Dactylococcopsis, Gloecapsa, Gloeothece,Merismopedia, Microcystis, Radiocystis, Rhabdoderma, Snowella,Synychococcus, Synechocystis, Thermosenechococcus, and Woronichinia;Nostacales Cyanobacteria from the genera Anabaena, Anabaenopsis,Aphanizomenon, Aulosira, Colothrix, Coleodesmium, Cyanospira,Cylindrospermosis, Cylindrospermum, Fremyella, Gleotrichia, Microchaete,Nodularia, Nostoc, Rexia, Richelia, Scytonema, Sprirestis, Toypothrix,Oscillatoriales; Cyanobacteria from the genera Arthrospira,Geitlerinema, Halomicronema, Halospirulina, Katagnymene, Leptolyngbya,Limnothrix, Lyngbya, Microcoleus, Oscillatoria, Phormidium,Planktothricoides, Planktothrix, Plectonema, Pseudoanabaena/Limnothrix,Schizothrix, Spirulina, Symploca, Trichodesmium, Tychonema;Pleurocapsales cyanobacterium from the genera Chroococcidiopsis,Dermocarpa, Dermocarpella, Myxosarcina, Pleurocapsa, Stanieria,Xenococcus, Prochlorophytes; Cyanobacterium from the genera Prochloron,Prochlorococcus, Prochlorothrix; and Stigonematales cyanobacterium fromthe genera Capsosira, Chlorogeoepsis, Fischerella, Hapalosiphon,Mastigocladopsis, Nostochopsis, Stigonema, Symphyonema, Symphonemopsis,Umezakia, and Westiellopsis. In certain embodiments, the Cyanobacteriumis from the genus Synechococcus, including, but not limited toSynechococcus bigranulatus, Synechococcus elongatus, Synechococcusleopoliensis, Synechococcus lividus, Synechococcus nidulans, andSynechococcus rubescens. In certain embodiments, the membrane-targetingdomain sequence is derived from a plasma membrane protein from S.elongatus sp. strain PCC7942 or Synechococcus sp. PCC 7002 (originallyknown as Agmenellum quadruplicatum).

In some embodiments, the membrane protein is a plasma membrane receptorprotein, such as a chemoreceptor or chemotaxis protein. Particularexamples include integral membrane chemoreceptors, e.g., transmembranechemoreceptors. Examples of chemoreceptors or chemotaxis proteinsinclude methyl-accepting chemotaxis proteins and amino acid chemotaxisreceptors, such as serine chemotaxis receptors (e.g., Tsr receptor fromEscherichia coli) and aspartate chemotaxis receptors. Themembrane-targeting domain can thus be obtained from the signal sequenceand/or transmembrane domains of any one or more of such bacterial plasmamembrane receptors.

In particular embodiments, the membrane-targeting domain is obtainedfrom (or derived from) a methyl-accepting chemotaxis protein (MCP). MCPscan be classified by topology type (see Zhulin, Adv Microb Physiol.45:157-198, 2001) and signaling domain class (see Alexander and Zhulin,PNAS USA. 104:2885-2890, 2007). Topology type I MCPs have largeperiplasmic ligand-binding domains and an elongated cytoplasmic regionconsisting of a HAMP domain (i.e., histidine kinases, adenylyl cyclases,methyl-binding proteins, and phosphatases) followed by a signalingdomain, which in turn is composed of “methylation,” “flexible bundle,”and “signaling” sub-domains (see Alexander and Zhulin, supra; andHazelbauer et al., Trends Biochem Sci. 33:9-19, 2008). MCPs clustertogether with other chemotaxis proteins in large arrays at the cellpole.

MCP arrays from variety of bacteria have been well-characterized,including, for example, E. coli, C. crescentus, Thermotoga maritima,Magnetospirillum magneticum, Rhodobacter sphaeroides, Treponemaprimitia, Listeria monocytogenes, Helicobacter hepaticus, Campylobacterjejuni, Acetonema longum, Borrella burgdorferi, Halothiobacillusneapolitanus, and Campylobacter jejuni (see Briegel et al., PNAS USA.106:17181-17186, 2009). The membrane-targeting domain can thus bederived from the signal sequence and/or transmembrane domains of an MCPfrom any one or more of these bacteria, or an MCP from any otherbacteria described herein or known in the art.

In certain embodiments, the MCP is encoded by PCC7942-0858 orPCC7942-1015 from S. elongatus. The polypeptide and polynucleotidesequence of the S. elongatus PCC7942-0858 MCP are set forth in SEQ IDNOS:199 and 200, respectively, and the polypeptide and polynucleotidesequence of the S. elongatus PCC7942-1015 MCP are set forth in SEQ IDNOS:201 and 202, respectively.

In some embodiments, the membrane-targeting domain comprises or consistsessentially of the N-terminal leader sequence, the first (N-terminal)transmembrane domain, and/or the second transmembrane domain ofPCC7942-0858, singly or in combination together. In certain instances,the bacterial membrane-targeting domain comprises or consistsessentially of about the N-terminal 43-53 amino acids of the MCP encodedby PCC7942-0858, for example, about residues 1-43, 4-44, 1-45, 1-46,1-47, 1-48, 1-49, 1-50, 1-51, 1-52, 1-53, 1-54, 1-55, 1-56, 1-57, 1-58,1-59, 1-60 of SEQ ID NO:199. In specific instances, the bacterialmembrane-targeting domain comprises or consists essentially of theN-terminal signal sequence and the two N-terminally proximal TMDs of theMCP encoded by PCC7942-0858, for example, about residues 1-43 of SEQ IDNO:199.

DGAT Polypeptides.

As used herein, a “diacylglycerol acyltransferase” (DGAT) polypeptideincludes any protein, polypeptide or peptide, obtainable from any cellsource, which demonstrates the ability to catalyze the production oftriacylglycerol from 1,2-diacylglycerol and fatty acyl substrates underenzyme reactive conditions, in addition to any naturally-occurring(e.g., allelic variants, orthologs) or non-naturally occurring variantsof a diacylglycerol acyltransferase sequence having such ability. DGATpolypeptides of the present disclosure also include bi-functionalproteins, such as those bi-functional proteins that exhibit a DGATactivity as well as a CoA:fatty alcohol acyltransferase activity, e.g.,a wax ester synthesis (WES) activity, as often found in many TAGproducing bacteria.

Diacylglycerol acyltransferases (DGATs) are members of theO-acyltransferase superfamily, which esterify either sterols ordiacylglycerols in an oleoyl-CoA-dependent manner. DGAT in particularesterifies diacylglycerols, which reaction represents the finalenzymatic step in the production of triacylglycerols in plants, fungiand mammals. Specifically, DGAT is responsible for transferring an acylgroup from acyl-coenzyme-A to the sn-3 position of 1,2-diacylglycerol(DAG) to form triacylglycerol (TAG). DGAT is an integral membraneprotein that has been generally described in Harwood (Biochem.Biophysics. Acta, 1301:7-56, 1996), Daum et al. (Yeast 16:1471-1510,1998), and Coleman et al. (Annu. Rev. Nutr. 20:77-103, 2000) (each ofwhich are herein incorporated by reference).

In plants and fungi, DGAT is associated with the membrane and lipid bodyfractions. In catalyzing TAGs, DGAT contributes mainly to the storage ofcarbon used as energy reserves. In animals, however, the role of DGAT ismore complex. DGAT not only plays a role in lipoprotein assembly and theregulation of plasma triacylglycerol concentration (Bell, R. M., etal.), but participates as well in the regulation of diacylglycerollevels (see Brindley, Biochemistry of Lipids, Lipoproteins andMembranes, eds. Vance, D. E. & Vance, J. E. (Elsevier, Amsterdam),171-203; and Nishizuka, Science 258:607-614, 1992, each of which areincorporated by reference).

In eukaryotes, at least three independent DGAT gene families (DGAT1,DGAT2, and PDAT) have been described that encode proteins with thecapacity to form TAG. Yeast contain all three of DGAT1, DGAT2, and PDAT,but the expression levels of these gene families varies during differentphases of the life cycle (Dahlqvst, A., et al. Proc. Natl. Acad. Sci.USA 97:6487-6492, 2000, incorporated by reference).

In prokaryotes, WS/DGAT from Acinetobacter calcoaceticus ADP1 representsthe first identified member of a widespread class of bacterial wax esterand TAG biosynthesis enzymes. This enzyme comprises a putativemembrane-spanning region but shows no sequence homology to the DGAT1 andDGAT2 families from eukaryotes. Under in vitro conditions, WS/DGAT showsa broad capability of utilizing a large variety of fatty alcohols, andeven thiols as acceptors of the acyl moieties of various acyl-CoAthioesters. WS/DGAT acyltransferase enzymes exhibit extraordinarilybroad substrate specificity. Genes for homologous acyltransferases havebeen found in almost all bacteria capable of accumulating neutrallipids, including, for example, Acinetobacter baylii, A. baumanii, andM. avium, and M. tuberculosis CDC1551, in which about 15 functionalhomologues are present (see, e.g., Daniel et al., J. Bacteriol.186:5017-5030, 2004; and Kalscheuer et al., J. Biol. Chem.287:8075-8082, 2003).

DGAT proteins may utilize a variety of acyl substrates in a host cell,including fatty acyl-CoA and fatty acyl-ACP molecules. In addition, theacyl substrates acted upon by DGAT enzymes may have varying carbon chainlengths and degrees of saturation, although DGAT may demonstratepreferential activity towards certain molecules.

Like other members of the eukaryotic O-acyltransferase superfamily,eukaryotic DGAT polypeptides typically contain a FYxDWWN (SEQ ID NO:15)heptapeptide retention motif, as well as a histidine (ortyrosine)-serine-phenylalanine (H/YSF) tripeptide motif, as described inZhongmin et al. (Journal of Lipid Research, 42:1282-1291, 2001) (hereinincorporated by reference). The highly conserved FYxDWWN (SEQ ID NO:15)is believed to be involved in fatty Acyl-CoA binding. In certaininstances, the DGAT polypeptide portion of the fusion proteins describedherein may thus comprise one or more these motifs.

DGAT polypeptides utilized according to the fusion proteins describedherein may be isolated from any organism, including eukaryotic andprokaryotic organisms. Eukaryotic organisms having a DGAT gene arewell-known in the art, and include various animals (e.g., mammals, fruitflies, nematodes), plants, parasites, and fungi (e.g., yeast such as S.cerevisiae and Schizosaccharomyces pombe). Examples of prokaryoticorganisms include certain actinomycetes, a group of Gram-positivebacteria with high G+C ratio, such as those from the representativegenera Actinomyces, Arthrobacter, Corynebacterium, Frankia, Micrococcus,Mocrimonospora, Mycobacterium, Nocardia, Propionibacterium, Rhodococcusand Streptomyces. Particular examples of actinomycetes that have one ormore genes encoding a DGAT activity include, for example, Mycobacteriumtuberculosis, M. avium, M. smegmatis, Micromonospora echinospora,Rhodococcus opacus, R. ruber, and Streptomyces lividans.

Additional examples of prokaryotic organisms that encode one or moreenzymes having a DGAT activity include members of the generaAcinetobacter, such as A. calcoaceticus, A. baumanii, A. baylii, andmembers of the genera Alcanivorax. In certain embodiments, a DGATpolypeptide is from Acinetobacter baylii sp. ADP1, a gram-negativetriglyceride forming prokaryote, which contains a well-characterizedDGAT (AtfA).

In particular embodiments, the DGAT polypeptide is an Acinetobacter DGAT(ADGAT), a Streptomyces DGAT, or an Alcanivorax DGAT. In certainembodiments, the DGAT polypeptide comprises or consists of a polypeptidesequence set forth in any one of SEQ ID NOs:58, 59, 60, or 61, or afragment or variant thereof. SEQ ID NO:58 is the sequence of DGATn; SEQID NO:59 is the sequence of Streptomyces coelicolor DGAT (ScoDGAT orSDGAT); SEQ ID NO:60 is the sequence of Alcanivorax borkumensis DGAT(AboDGAT); and SEQ ID NO:61 is the sequence of DGATd.

In certain embodiments, the modified photosynthetic microorganisms ofthe present disclosure may express two or more intracellularlocalization domain-DGAT fusion proteins. The DGAT polypeptides may bythe same or different. In particular embodiments, the followingintracellular localization domain-DGAT fusions are co-expressed inmodified photosynthetic microorganisms, e.g., Cyanobacteria, using oneof the following double DGAT strains: ADGATd::ScoDGAT;ADGATd(NS1)::ADGATd(NS2); ADGATn(NS1)::ADGATn(NS2);ADGATn(NS1)::SDGAT(NS2); SDGAT(NS1)::ADGATn(NS2);SDGAT(NS1)::SDGAT(NS2). For the NS1 vector, pAM2291, EcoRI follows ATGand is part of the open reading frame (ORF). For the NS2 vector,pAM1579, EcoRI follows ATG and is part of the ORF. A DGAT having EcoRInucleotides following ATG may be cloned in either pAM2291 or pAM1579;such a DGAT is referred to as ADGATd. Other embodiments utilize thevector, pAM2314FTrc3, which is an NS1 vector with Nde/BglII sites, orthe vector, pAM1579FTrc3, which is the NS2 vector with Nde/BglII sites.A DGAT without EcoRI nucleotides may be cloned into either of these lasttwo vectors. Such a DGAT is referred to as ADGATn. Modifiedphotosynthetic microorganisms expressing different DGATs express TAGshaving different fatty acid compositions. Accordingly, certainembodiments contemplate expressing two or more different intracellularlocalization domain-DGAT fusions, in order to produce TAGs having variedfatty acid compositions.

Peptide Linkers.

In certain embodiments, a peptide linker sequence may be employed toseparate the DGAT polypeptide(s) and the heterologous intracellularlocalization domain(s) by a distance sufficient to ensure that eachpolypeptide folds into its desired secondary and tertiary structures.Such a peptide linker sequence can be incorporated into the fusionprotein using standard techniques well known in the art.

Certain peptide linker sequences may be chosen based on the followingexemplary factors: (1) their ability to adopt a flexible extendedconformation; (2) their inability to adopt a secondary structure thatcould interact with functional epitopes on the first and secondpolypeptides; (3) their physiological stability; and (4) the lack ofhydrophobic or charged residues that might react with the polypeptidefunctional epitopes, or other features. See, e.g., George and Heringa, JProtein Eng. 15:871-879, 2002.

The linker sequence can be essentially any length, but is generally fromabout 1 to about 300 amino acids in length. Particular linkers can havean overall amino acid length of about 1-300 amino acids, 1-250, 1-200amino acids, 1-150 amino acids, 1-100 amino acids, 1-90 amino acids,1-80 amino acids, 1-70 amino acids, 1-60 amino acids, 1-50 amino acids,1-40 amino acids, 1-30 amino acids, 1-20 amino acids, 1-10 amino acids,1-5 amino acids, 1-4 amino acids, 1-3 amino acids, or about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120, 130,140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270,280, 290, 300 or more amino acids in length.

Certain amino acid sequences which may be usefully employed as linkersinclude those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphyet al., PNAS USA. 83:8258-8262, 1986; U.S. Pat. Nos. 4,935,233 and4,751,180. Particular peptide linker sequences contain Gly, Ser, and/orAsn residues. Other near neutral amino acids, such as Thr and Ala mayalso be employed in the peptide linker sequence, if desired.

Certain exemplary linkers include Gly, Ser and/or Asn-containinglinkers, as follows: [G]_(x), [S]_(x), [N]_(x), [GS]_(x), [GGS]_(x),[GSS]_(x), [GSGS]_(x) (SEQ ID NO:203), [GGSG]_(x) (SEQ ID NO:204),[GGGS]_(x) (SEQ ID NO:205), [GGGGS]_(x) (SEQ ID NO:206), [GN]_(x),[GGN]_(x), [GNN]_(x), [GNGN]_(x)(SEQ ID NO:207), [GGNG]_(x) (SEQ IDNO:208), [GGGN]_(x) (SEQ ID NO:209), [GGGGN]_(x) (SEQ ID NO:210)linkers, where _(x) is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 25, 30, 40, 50 or more. Other combinations ofthese and related amino acids will be apparent to persons skilled in theart.

In certain embodiments, however, any one or more of the peptide linkersare optional. For instance, linker sequences may not required when thebacterial membrane protein (from which the membrane- or PM-targetingdomain is derived) and the DGAT polypeptides have non-essential regions(e.g., N-terminal and/or C-terminal amino acids, or for the plasmamembrane proteins, regions just downstream of the signal sequencesand/or transmembrane domains) that can be used to separate thefunctional domains and prevent steric interference.

(ii) lipid Biosynthesis Proteins

In various embodiments, modified photosynthetic microorganisms of thepresent disclosure further comprise one or more exogenous (e.g.,introduced) or overexpressed nucleic acids that encode a lipidbiosynthesis protein, e.g., a polypeptide having an activity associatedwith triglyceride biosynthesis or fatty acid biosynthesis, including butnot limited to any of those described herein. In particular instances, amodified photosynthetic microorganism may comprise reduced expressionand/or activity of one or more selected lipid biosynthesis proteins.Certain of these proteins are described in greater detail below.

In particular embodiments, the exogenous nucleic acid does not comprisea nucleic acid sequence that is native to the microorganism's genome. Insome embodiments, the exogenous nucleic acid comprises a nucleic acidsequence that is native to the microorganism's genome, but it has beenintroduced into the microorganism, e.g., in a vector or by molecularbiology techniques, for example, to increase expression of the nucleicacid and/or its encoded polypeptide in the microorganism. In certainembodiments, the expression of a native or endogenous nucleic acid andits corresponding protein can be increased by introducing a heterologouspromoter upstream of the native gene. As noted above, lipid biosynthesisproteins can be involved in triglyceride biosynthesis, fatty acidsynthesis, wax ester synthesis, or any combination thereof.

Triglyceride Biosynthesis.

Triglycerides, or triacylglycerols (TAGs), consist primarily of glycerolesterified with three fatty acids, and yield more energy upon oxidationthan either carbohydrates or proteins. Triglycerides provide animportant mechanism of energy storage for most eukaryotic organisms. Inmammals, TAGs are synthesized and stored in several cell types,including adipocytes and hepatocytes (Bell et al. Annu. Rev. Biochem.49:459-487, 1980) (incorporated by reference). In plants, TAG productionis mainly important for the generation of seed oils.

In contrast to eukaryotes, the observation of triglyceride production inprokaryotes has been limited to certain actinomycetes, such as membersof the genera Mycobacterium, Nocardia, Rhodococcus and Streptomyces, inaddition to certain members of the genus Acinetobacter. In certainActinomycetes species, triglycerides may accumulate to nearly 80% of thedry cell weight, but accumulate to only about 15% of the dry cell weightin Acinetobacter. In general, triglycerides are stored in sphericallipid bodies, with quantities and diameters depending on the respectivespecies, growth stage, and cultivation conditions. For example, cells ofRhodococcus opacus and Streptomyces lividans contain only few TAGs whencultivated in complex media with a high content of carbon and nitrogen;however, the lipid content and the number of TAG bodies increasedrastically when the cells are cultivated in mineral salt medium with alow nitrogen-to-carbon ratio, yielding a maximum in the late stationarygrowth phase. At this stage, cells can be almost completely filled withlipid bodies exhibiting diameters ranging from 50 to 400 nm. One exampleis R. opacus PD630, in which lipids can reach more than 70% of the totalcellular dry weight.

In bacteria, TAG formation typically starts with the docking of adiacylglycerol acyltransferase enzyme to the plasma membrane, followedby formation of small lipid droplets (SLDs). These SLDs are only somenanometers in diameter and remain associated with the membrane-dockedenzyme. In this phase of lipid accumulation, SLDs typically form anemulsive, oleogenous layer at the plasma membrane. During prolongedlipid synthesis, SLDs leave the membrane-associated acyltransferase andconglomerate to membrane-bound lipid prebodies. These lipid prebodiesreach distinct sizes, e.g., about 200 nm in A. calcoaceticus and about300 nm in R. opacus, before they lose contact with the membrane and arereleased into the cytoplasm. Free and membrane-bound lipid prebodiescorrespond to the lipid domains occurring in the cytoplasm and at thecell wall, as observed in M. smegmatis during fluorescence microscopyand also confirmed in R. opacus PD630 and A. calcoaceticus ADP1 (see,e.g., Christensen et al., Mol. Microbiol. 31:1561-1572, 1999; andWältermann et al., Mol. Microbiol. 55:750-763, 2005). Inside the lipidprebodies, SLDs coalesce with each other to form the homogenous lipidcore found in mature lipid bodies, which often appear opaque in electronmicroscopy.

The compositions and structures of bacterial TAGs vary considerablydepending on the microorganism and on the carbon source. In addition,unusual acyl moieties, such as phenyldecanoic acid and 4,8,12 trimethyltridecanoic acid, may also contribute to the structural diversity ofbacterial TAGs (see, e.g., Alvarez et al., Appl Microbiol Biotechnol.60:367-76, 2002).

As with eukaryotes, the main function of TAGs in prokaryotes is to serveas a storage compound for energy and carbon. TAGs, however, may provideother functions in prokaryotes. For example, lipid bodies may act as adeposit for toxic or useless fatty acids formed during growth onrecalcitrant carbon sources, which must be excluded from the plasmamembrane and phospholipid (PL) biosynthesis. Furthermore, manyTAG-accumulating bacteria are ubiquitous in soil, and in this habitat,water deficiency causing dehydration is a frequent environmental stress.Storage of evaporation-resistant lipids might be a strategy to maintaina basic water supply, since oxidation of the hydrocarbon chains of thelipids under conditions of dehydration would generate considerableamounts of water. Cyanobacteria such as Synechococcus, however, do notproduce triglycerides, because these organisms lack the enzymesnecessary for triglyceride biosynthesis.

Triglycerides are synthesized from fatty acids and glycerol. As onemechanism of triglyceride (TAG) synthesis, sequential acylation ofglycerol-3-phosphate via the “Kennedy Pathway” leads to the formation ofphosphatidate. Phosphatidate is then dephosphorylated by the enzymephosphatidate phosphatase to yield 1,2 diacylglycerol (DAG). Using DAGas a substrate, at least three different classes of enzymes are capableof mediating TAG formation. As one example, an enzyme havingdiacylglycerol acyltransferase (DGAT) activity catalyzes the acylationof DAG using acyl-CoA as a substrate. Essentially, DGAT enzymes combineacyl-CoA with 1,2 diacylglycerol molecule to form a TAG. As analternative, Acyl-CoA-independent TAG synthesis may be mediated by aphospholipid:DAG acyltransferase found in yeast and plants, which usesphospholipids as acyl donors for DAG esterification. Third, TAGsynthesis in animals and plants may be mediated by aDAG-DAG-transacylase, which uses DAG as both an acyl donor and acceptor,yielding TAG and monoacylglycerol.

Modified photosynthetic microorganisms, e.g., Cyanobacteria, of thepresent disclosure may comprise one or more exogenous polynucleotidesencoding polypeptides comprising one or more of the polypeptides andenzymes described herein.

Since wild type Cyanobacteria do not typically encode the enzymesnecessary for triglyceride synthesis, such as the enzymes havingdiacylglycerol acyltransferase activity, embodiments of the presentdisclosure include genetically modified Cyanobacteria that comprisepolynucleotides encoding one or more DGAT fusion proteins, optionally incombination with one or more enzymes having a fatty acyl-CoA synthetaseactivity. In particular embodiments, the one or more exogenouspolynucleotides encode a DGAT fusion protein described herein and afatty acyl-CoA synthetase, or a functional variant or fragment thereof.

Moreover, since triglycerides are typically formed from fatty acids, thelevel of fatty acid biosynthesis in a cell may limit the production oftriglycerides. Increasing the level of fatty acid biosynthesis may,therefore, allow increased production of triglycerides. As discussedbelow, acetyl-CoA carboxylase catalyzes the commitment step to fattyacid biosynthesis. Thus, certain embodiments of the present disclosureinclude Cyanobacterium, and methods of use thereof, comprisingpolynucleotides that encode one or more enzymes having acetyl-CoAcarboxylase activity to increase fatty acid biosynthesis and lipidproduction, in addition to one or more DGAT fusion proteins andoptionally one or more enzymes having fatty acyl-CoA synthetaseactivity, to catalyze triglyceride production. These and relatedembodiments are detailed below.

Fatty Acid Biosynthesis.

Fatty acids are a group of negatively charged, linear hydrocarbon chainsof various length and various degrees of oxidation states. The negativecharge is located at a carboxyl end group and is typically deprotonatedat physiological pH values (pK˜2-3). The length of the fatty acid ‘tail’determines its water solubility (or rather insolubility) and amphipathiccharacteristics. Fatty acids are components of phospholipids andsphingolipids, which form part of biological membranes, as well astriglycerides, which are primarily used as energy storage moleculesinside cells.

Fatty acids are formed from acetyl-CoA and malonyl-CoA precursors.Malonyl-CoA is a carboxylated form of acetyl-CoA, and contains a3-carbon dicarboxylic acid, malonate, bound to Coenzyme A. Acetyl-CoAcarboxylase catalyzes the 2-step reaction by which acetyl-CoA iscarboxylated to form malonyl-CoA. In particular, malonate is formed fromacetyl-CoA by the addition of CO₂ using the biotin cofactor of theenzyme acetyl-CoA carboxylase.

Fatty acid synthase (FAS) carries out the chain elongation steps offatty acid biosynthesis. FAS is a large multienzyme complex. In mammals,FAS contains two subunits, each containing multiple enzyme activities.In bacteria and plants, individual proteins, which associate into alarge complex, catalyze the individual steps of the synthesis scheme.For example, in bacteria and plants, the acyl carrier protein is asmaller, independent protein.

Fatty acid synthesis starts with acetyl-CoA, and the chain grows fromthe “tail end” so that carbon 1 and the alpha-carbon of the completefatty acid are added last. The first reaction is the transfer of anacetyl group to a pantothenate group of acyl carrier protein (ACP), aregion of the large mammalian fatty acid synthase (FAS) protein. In thisreaction, acetyl CoA is added to a cysteine —SH group of the condensingenzyme (CE) domain: acetyl CoA+CE-cys-SH->acetyl-cys-CE+CoASH.Mechanistically, this is a two step process, in which the group is firsttransferred to the ACP (acyl carrier peptide), and then to the cysteine—SH group of the condensing enzyme domain.

In the second reaction, malonyl CoA is added to the ACP sulfhydrylgroup: malonyl CoA+ACP-SH->malonyl ACP+CoASH. This —SH group is part ofa phosphopantethenic acid prosthetic group of the ACP.

In the third reaction, the acetyl group is transferred to the malonylgroup with the release of carbon dioxide: malonylACP+acetyl-cys-CE->beta-ketobutyryl-ACP+CO₂.

In the fourth reaction, the keto group is reduced to a hydroxyl group bythe beta-ketoacyl reductase activity:beta-ketobutyryl-ACP+NADPH+H⁺->beta-hydroxybutyryl-ACP+NAD⁺.

In the fifth reaction, the beta-hydroxybutyryl-ACP is dehydrated to forma trans-monounsaturated fatty acyl group by the beta-hydroxyacyldehydratase activity: beta-hydroxybutyryl-ACP->2-butenoyl-ACP+H₂O.

In the sixth reaction, the double bond is reduced by NADPH, yielding asaturated fatty acyl group two carbons longer than the initial one (anacetyl group was converted to a butyryl group in this case):2-butenoyl-ACP+NADPH+H⁺->butyryl-ACP+NADP⁺. The butyryl group is thentransferred from the ACP sulfhydryl group to the CE sulfhydryl:butyryl-ACP+CE-cys-SH->ACP-SH+butyryl-cys-CE. This step is catalyzed bythe same transferase activity utilized previously for the originalacetyl group. The butyryl group is now ready to condense with a newmalonyl group (third reaction above) to repeat the process. When thefatty acyl group becomes 16 carbons long, a thioesterase activityhydrolyses it, forming free palmitate:palmitoyl-ACP+H₂O->palmitate+ACP-SH. Fatty acid molecules can undergofurther modification, such as elongation and/or desaturation.

Modified photosynthetic microorganisms, e.g., Cyanobacteria, maycomprise one or more exogenous polynucleotides encoding any of the abovepolypeptides or enzymes involved in fatty acid synthesis. In particularembodiments, the enzyme is an acetyl-CoA carboxylase or a variant orfunctional fragment thereof.

Wax Ester Synthesis.

Wax esters are esters of a fatty acid and a long-chain alcohol. Theseneutral lipids are composed of aliphatic alcohols and acids, with bothmoieties usually long-chain (e.g., C₁₆ and C₁₈) or very-long-chain (C₂₀and longer) carbon structures, though medium-chain-containing wax estersare included (e.g., C₁₀, C₁₂ and C₁₄). Wax esters have diversebiological functions in bacteria, insects, mammals, and terrestrialplants and are also important substrates for a variety of Industrialapplications. Various types of wax ester are widely used in themanufacture of fine chemicals such as cosmetics, candles, printing inks,lubricants, coating stuffs, and others.

In certain organisms, such as Acinetobacter, the pathway for wax estersynthesis of Acinetobacter spp. has been assumed to start from acylcoenzyme A (acyl-CoA), which is then reduced to the correspondingalcohol via acyl-CoA reductase and aldehyde reductase. In otherorganisms, for example, wax ester biosynthesis involves elongation ofsaturated C₁₆ and C₁₈ fatty acyl-CoAs to very-long-chain fatty acid waxprecursors between 24 and 34 carbons in length, and their subsequentmodification by either the alkane-forming (decarbonylation) or thealcohol-forming (acyl reduction) pathway (see Li et al., PlantPhysiology 148:97-107, 2008).

In certain aspects, wax ester synthesis can occur via the acyl-ACP->acylaldehyde pathway. In this pathway, acyl-ACP reductase overexpressionincreases conversion of acyl-ACP into acyl aldehydes, alcoholdehydrogenase overexpression then increases conversion of acyl aldehydesinto fatty alcohols, and DGAT overexpression cooperatively increasesconversion of the fatty alcohols into their corresponding wax esters.Modified photosynthetic microorganisms, e.g., Cyanobacteria, maytherefore comprise one or more exogenous polynucleotides encoding any ofthe above polypeptides or enzymes involved in wax ester synthesis.

Acyl Carrier Proteins.

Embodiments of the present disclosure optionally include one or moreexogenous (e.g., recombinantly introduced) or overexpressed ACPproteins. These proteins play crucial roles in fatty acid synthesis.Fatty acid synthesis in bacteria, including Cyanobacteria, is carriedout by highly conserved enzymes of the type II fatty acid synthasesystem (FAS II; consisting of about 19 genes) in a sequential, regulatedmanner. Acyl carrier protein (ACP) plays a central role in this processby carrying all the intermediates as thioesters attached to the terminusof its 4′-phosphopantetheine prosthetic group (ACP-thioesters). Apo-ACP,the product of acp gene, is typically activated by a phosphopantetheinyltransferase (PPT) such as the acyl carrier protein synthase (AcpS) typefound in E. coli or the Sfp (surfactin type) PPT as characterized inBacillus subtilis. Cyanobacteria possess an Sfp-like PPT, which isunderstood to act in both primary and secondary metabolism. Embodimentsof the present disclosure therefore include overexpression of PPTs suchas AcpS and/or Sfp-type PPTs in combination with overexpression ofcognate ACP encoding genes, such as ACP.

The ACP-thioesters are substrates for all of the enzymes of the FAS IIsystem. The end product of fatty acid synthesis is a long acyl chaintypically consisting of about 14-18 carbons attached to ACP by athioester bond.

At least three enzymes of the FAS II system in other bacteria can besubject to feedback inhibition by acyl-ACPs: 1) the ACCase complex—aheterotetramer of the AccABCD genes that catalyzes the production ofmalonyl-coA, the first step in the pathway; 2) the product of the FabHgene (β-ketoacyl-ACP synthase III), which catalyzes the condensation ofacetyl-CoA with malonyl-ACP; and 3) the product of the Fabl gene(enoyl-ACP reductase), which catalyzes the final elongation step in eachround of elongation. Certain proteins such as acyl-ACP reductase arecapable of increasing fatty acid production in photosynthetic bacteriasuch as Cyanobacteria, and it is believed that overexpression of ACP incombination with this protein and possibly other biosynthesis proteinswill further increases fatty acid production in such strains.

An ACP can be derived from a variety of eukaryotic organisms,microorganisms (e.g., bacteria, fungi), or plants. In certainembodiments, an ACP polynucleotide sequence and its correspondingpolypeptide sequence are derived from Cyanobacteria such asSynechococcus. In certain embodiments, ACPs can be derived from plantssuch as spinach. SEQ ID NOS:5-12 provide the nucleotide and polypeptidesequences of exemplary bacterial ACPs from Synechococcus andAcinetobacter, and SEQ ID NOS:13-14 provide the same for an exemplaryplant ACP from Spinacia oleracea (spinach). SEQ ID NOS:5 and 6 derivefrom Synechococcus elongatus PCC7942, and SEQ ID NOS:7-12 derive fromAcinetobacter sp. ADP1.

Examples of prokaryotic organisms having an ACP Include certainactinomycetes, a group of Gram-positive bacteria with high G+C ratio,such as those from the representative genera Actinomyces, Arthrobacter,Corynebacterium, Frankia, Micrococcus, Mocrimonospora, Mycobacterium,Nocardia, Propionibacterium, Rhodococcus and Streptomyces. Particularexamples of actinomycetes that have one or more genes encoding an ACPactivity include, for example, Mycobacterium tuberculosis, M. avium, M.smegmatis, Micromonospora echinospora, Rhodococcus opacus, R. ruber, andStreptomyces lividans. Additional examples of prokaryotic organisms thatencode one or more enzymes having an ACP activity include members of thegenera Acinetobacter, such as A. calcoaceticus, A. baumanii, A. baylii,and members of the genera Alcanivorax. In certain embodiments, an ACPgene or enzyme is isolated from Acinetobacter baylii sp. ADP1, agram-negative triglyceride forming prokaryote.

Acyl ACP Synthases (Aas).

Acyl-ACP synthetases (Aas) catalyze the ATP-dependent acylation of thethiol of acyl carrier protein (ACP) with fatty acids, including thosefatty acids having chain lengths from about C4 to C18. In Cyanobacteria,among other functions, Aas enzymes not only directly incorporateexogenous fatty acids from the culture medium into other lipids, butalso play a role in the recycling of acyl chains from lipid membranes.Deletion of Aas in cyanobacteria can lead to secretion of free fattyacids into the culture medium. See, e.g., Kaczmarzyk and Fulda, PlantPhysiology 152:1598-1610, 2010.

Certain embodiments may overexpress one or more Aas polypeptidesdescribed herein and known in the art. According to one non-limitingtheory, overexpression of Aas in combination with overexpression of ACPleads to increased TAG production in DGAT-expressing strains, forexample, by boosting acyl-ACP levels. Overexpression of Aas in optionalcombination with overexpression of ACP may likewise increase wax esterformation, for example, when combined with overexpression of one or morealcohol dehydrogenase(s) and wax ester synthase(s), such as abi-functional DGAT. Certain embodiments therefore include modifiedphotosynthetic microorganisms comprising overexpressed Aaspolypeptide(s), optionally in combination with overexpressed ACPpolypeptide(s), especially when combined with overexpression of alcoholdehydrogenase, acyl-ACP reductase (e.g., orf1594), and wax estersynthase (e.g., aDGAT).

Examples of bacterial Aas enzymes include those derived from E. coli,Acinetobacter, and Vibrio sp. such as V. harveyi (see, e.g., Shanklln,Protein Expression and Purification. 18:355-360, 2000; Jiang et al.,Biochemistry. 45:10008-10019, 2006). SEQ ID NOS:43 and 44, respectively,provide the nucleotide and polypeptide sequences of an exemplary Aasfrom Synechococcus elongatus PCC 7942 (0918).

In certain embodiments, the Aas is derived from the same organism as theoverexpressed ACP, DGAT, and/or the TES, if any one of thesepolypeptides is employed in combination with an Aas. Accordingly,certain embodiments include Aas sequences from any of the organismsdescribed herein for deriving a DGAT or TES, including, for example,various animals (e.g., mammals, fruit flies, nematodes), plants,parasites, and fungi (e.g., yeast such as S. cerevisiae andSchizosaccharomyces pombe). Examples of prokaryotic organisms includecertain actinomycetes, a group of Gram-positive bacteria with high G+Cratio, such as those from the representative genera Actinomyces,Arthrobacter, Corynebacterium, Frankia, Micrococcus, Mocrimonospora,Mycobacterium, Nocardia, Propionibacterium, Rhodococcus andStreptomyces. Particular examples of actinomycetes that have one or moregenes encoding an Aas activity include, for example, Mycobacteriumtuberculosis, M. avium, M. smegmatis, Micromonospora echinospora,Rhodococcus opacus, R. ruber, and Streptomyces lividans. Additionalexamples of prokaryotic organisms that encode one or more enzymes havingan Aas activity include members of the genera Acinetobacter, such as A.calcoaceticus, A. baumanii, A. baylii, and members of the generaAlcanivorax. In certain embodiments, an Aas gene or enzyme is isolatedfrom Acinetobacter baylii sp. ADP1, a gram-negative triglyceride formingprokaryote.

According to one non-limiting theory, an endogenous aldehydedehydrogenase may be acting on the excess acyl-aldehydes generated byoverexpressed orf1594 and converting them to free fatty acids. Thenormal role of such a dehydrogenase might involve removing or otherwisedealing with damaged lipids. In this scenario, it is then likely thatthe Aas gene product recycles these free fatty acids by ligating them toACP. Accordingly, reducing or eliminating expression of the Aas geneproduct might ultimately increase production of fatty acids, by reducingor preventing their transfer to ACP. Hence, certain aspects includemutations (e.g., genomic) such as point mutations or insertions thatreduce or eliminate the enzymatic activity of one or more endogenousacyl-ACP synthetases (or synthases). Also included are full or partialdeletions of an endogenous gene encoding an Aas protein.

Acy-ACP Reductases.

Acyl-ACP reductases (or acyl-ACP dehydrogenases) are members of thereductase or short-chain dehydrogenase family, and are key enzymes ofthe type II fatty acid synthesis (FAS) system. Among other potentialcatalytic activities, an “acyl-ACP reductase” or “acyl-ACPdehydrogenase” as used herein is capable of catalyzing the conversion(reduction) of acyl-ACP to an acyl aldehyde (see Schirmer et al., supra)and the concomitant oxidation of NAD(P)H to NADP⁺. In some embodiments,the acyl-ACP reductase preferentially interacts with acyl-ACP, and doesnot interact significantly with acyl-CoA, i.e., it does notsignificantly catalyze the conversion of acyl-CoA to acyl aldehyde.

Acyl-ACP reductases can be derived from a variety of plants andbacteria, included photosynthetic microorganisms such as Cyanobacteria.One exemplary acyl-ACP reductase is encoded by orf1594 of Synechococcuselongatus PCC7942 (see SEQ ID NOs:1 and 2 for the polynucleotide andpolypeptide sequences, respectively). Another exemplary acyl-ACPreductase is encoded by orfsll0209 of Synechocystis sp. PCC6803 (SEQ IDNOs:3 and 4 for the polynucleotide and polypeptide sequences,respectively).

Alcohol Dehydrogenases.

Embodiments of the present disclosure optionally include one or morealcohol dehydrogenase polypeptides. Examples of alcohol dehydrogenasesinclude those capable of using acyl or fatty aldehydes (e.g., one ormore of nonyl-aldehyde, C₁₂, C₁₄, C₁₆, C₁₈, C₂₀ fatty aldehyde) as asubstrate, and converting them into fatty alcohols. Specific examplesinclude long-chain alcohol dehydrogenases, capable of using long-chainaldehydes (e.g., C₁₆, C₁₈, C₂₀) as substrates. In certain embodiments,the alcohol dehydrogenase is naturally-occurring or endogenous to themodified microorganism, and is sufficient to convert increased acylaldehydes (produced by acyl-ACP reductase) into fatty alcohols, andthereby contribute to increased wax ester production and overallsatisfactory growth characteristics. In certain embodiments, the alcoholdehydrogenase is derived from a microorganism that differs from the onebeing modified.

In these and related embodiments, expression or overexpression of analcohol dehydrogenase may increase shunting of acyl aldehydes towardsproduction of fatty alcohols, and away from production of other productssuch as alkanes, fatty acids, or triglycerides. When combined with oneor more wax ester synthases, such as DGAT or other enzyme having waxester synthase activity (e.g., the ability to convert fatty alcoholsinto wax esters), alcohol dehydrogenases may contribute to production ofwax esters. They may also reduce accumulation of potentially toxic acylaldehydes, and thereby improve growth characteristics of a modifiedmicroorganism.

Non-limiting examples of alcohol dehydrogenases include those encoded byslr1192 of Synechocystis sp. PCC6803 (SEQ ID NOS:104-105) and ACIAD3612of Acinetobacter baylyi (SEQ ID NOS:106-107). Also included are homologsor paralogs thereof, functional equivalents thereof, and fragments orvariants thereofs. Functional equivalents can include alcoholdehydrogenases with the ability to efficiently convert acyl aldehydes(e.g., C₆, C₈, C₁₀, C₁₂, C₁₄, C₁₆, C₁₈, C₂₀ aldehydes) into fattyalcohols. Specific examples of functional equivalents include long-chainalcohol dehydrogenases, having the ability to utilize long-chainaldehydes (e.g., C₁₆, C₁₈, C₂₀) as substrates.

In particular embodiments, the alcohol dehydrogenase has the amino acidsequence of SEQ ID NO:105 (encoded by the polynucleotide sequence of SEQID NO:104), or an active fragment or variant of this sequence. In someembodiments, the alcohol dehydrogenase has the amino acid sequence ofSEQ ID NO:107 (encoded by the polynucleotide sequence of SEQ ID NO:106),or an active fragment or variant of this sequence.

Aldehyde Dehydrogenases.

Embodiments of the present disclosure optionally include one or moreoverexpressed or introduced aldehyde dehydrogenases. Examples ofaldehyde dehydrogenases include enzymes capable of using acyl aldehydes(e.g., nonyl-aldehyde, C16 fatty aldehyde) as a substrate, andconverting them into fatty acids. In certain embodiments, the aldehydedehydrogenase is naturally-occurring or endogenous to the modifiedmicroorganism, and is sufficient to convert increased acyl aldehydes(produced by acyl-ACP reductase) into fatty acids, and therebycontribute to increased fatty acid production and overall satisfactorygrowth characteristics.

In certain embodiments, the aldehyde dehydrogenase can be overexpressed,for example, by recombinantly introducing a polynucleotide that encodesthe enzyme, increasing expression of an endogenous enzyme, or both. Analdehyde dehydrogenase can be overexpressed in a strain that alreadyexpresses a naturally-occurring or endogenous enzyme, to furtherincrease fatty acid production of an acyl-ACP reductase over-expressingstrain and/or improve its growth characteristics, relative, for example,to an acyl-ACP reductase-overexpressing strain that only expressesendogenous aldehyde dehydrogenase. An aldehyde dehydrogenase can also beexpressed or overexpressed in a strain that does not have a naturallyoccurring aldehyde dehydrogenase of that type, e.g., it does notnaturally express an enzyme that is capable of efficiently convertingacyl aldehydes such as nonyl-aldehyde into fatty acids.

In these and related embodiments, expression or overexpression of analdehyde dehydrogenase may increase shunting of acyl aldehydes towardsproduction of fatty acids, and away from production of other productssuch as alkanes. It may also reduce accumulation of potentially toxicacyl aldehydes, and thereby improve growth characteristics of a modifiedmicroorganism.

One exemplary aldehyde dehydrogenase is encoded by orf0489 ofSynechococcus elongatus PCC7942. Also included are homologs or paralogsthereof, functional equivalents thereof, and fragments or variantsthereof. Functional equivalents can include aldehyde dehydrogenases withthe ability to efficiently convert acyl aldehydes (e.g., nonyl-aldehyde)into fatty acids. In certain embodiments, the aldehyde dehydrogenase hasthe amino acid sequence of SEQ ID NO:103 (encoded by the polynucleotidesequence of SEQ ID NO:102), or an active fragment or variant of thissequence.

Particular embodiments include photosynthetic microorganisms havingreduced expression and/or activity of one or more aldehydedehydrogenases, for instance, in the production of wax esters. Includedare mutations (e.g., genomic) that reduce or eliminate the enzymaticactivity of one or more endogenous aldehyde dehydrogenases, such aspoint mutations, insertions, or full or partial deletion mutations.Certain embodiments include a modified Synechococcus elongatus PCC7942having a full or partial deletion of orf0489.

Aldehyde Decarbonylases.

Certain embodiments include photosynthetic microorganisms having reducedexpression and/or activity of one or more aldehyde decarbonylases. Asused herein, an “aldehyde decarbonylase” is capable of catalyzing theconversion of an acyl aldehyde (or fatty aldehyde) to an alkane oralkene. Included are members of the ferritin-like or ribonucleotidereductase-like family of nonheme diiron enzymes (see, e.g., Stubbe etal., Trends Biochem Sci. 23:438-43, 1998).

According to one non-limiting theory, because the aldehyde decarbonylaseencoded by PCC7942_orf1593 (from Synechococcus) or PCC6803_orfsll0208(from Synechostis sp. PCC6803) utilizes acyl aldehyde as a substrate foralkane or alkene production, reducing expression of this protein mayfurther increase yields of free fatty acids by shunting acyl aldehydes(produced by acyl-ACP reductase) away from an alkane-producing pathway,and towards a fatty acid-producing pathway. PCC7942_orf1593 andPCC6803_orfsll0208 orthologs can be found, for example, in N.punctiforme PCC73102, Thermosynechococcus elongatus BP-1, Synechococcussp. Ja-3-3AB, P. marinus MIT9313, P. marinus NATL2A, and Synechococcussp. RS 9117, the latter having at least two paralogs (RS 9117-1 and -2).

Particular embodiments include mutations (e.g., genomic) that reduce oreliminate the enzymatic activity of one or more endogenous aldehydedecarbonylases, for instance, in the production of fatty acids or waxesters, optionally in combination with reduced expression of one or moreendogenous aldehyde dehydrogenases. Also included are point mutations,insertions, and full or partial deletions of an endogenous gene encodingan aldehyde decarbonylase. Certain embodiments include a modifiedSynechococcus elongatus PCC7942 having a full or partial deletion oforf1593.

Thioesterases.

Certain embodiment include one or more exogenous or overexpressedthioesterase enzymes, optionally in combination with at least one of anintroduced ACP enzyme, an introduced Aas enzyme, or both. For instance,one embodiment relates to the use an introduced ACP and/or Aas toincrease the growth and/or fatty acid production of a free fatty acidproducing TES strain, such as a TesA strain or a FatB strain (i.e., astrain having an introduced TesA or FatB). Thioesterases, as referred toherein, exhibit esterase activity (splitting of an ester into acid andalcohol, in the presence of water) specifically at a thiol group. Fattyacids are often attached to cofactor molecules, such as coenzyme A (CoA)and acyl carrier protein (ACP), by thioester linkages during the processof de novo fatty acid synthesis. Certain embodiments employthioesterases having acyl-ACP thioesterase activity, acyl-CoAthioesterase activity, or both activities. Examples of thioesteraseshaving both activities (i.e., acyl-ACP/acyl-CoA thioesterases) includeTesA and related embodiments. In certain embodiments, a selectedthioesterase has acyl-ACP thioesterase activity but not acyl-CoAthioesterase activity. Examples of thioesterases having only acyl-ACPthioesterase activity include the FatB thioesterases and relatedembodiments.

Certain thioesterases have both thioesterase activity andlysophospholipase activity. Specific examples of thioesterases includeTesA, TesB, and related embodiments. Certain embodiments may employperiplasmically-localized or cytoplasmically-localized enzymes thatthioesterase activity, such as E. coli TesA or E. coli TesB. Forinstance, wild type TesA, being localized to the periplasm, is normallyused to hydrolyze thioester linkages of fatty acid-ACP (acyl-ACP) orfatty acid-CoA (acyl-CoA) compounds scavenged from the environment. Amutant thioesterase, PldC (referred to interchangeably as PldC/*TesA or*TesA), is not exported to the periplasm due to deletion of anN-terminal amino acid sequence required for proper transport of TesAfrom the cytoplasm to the periplasm. This deletion results in acytoplasmic-localized PldC(*TesA) protein that has access to endogenousacyl-ACP and acyl-CoA intermediates. Other mutations or deletions in theN-terminal region of TesA can be used to achieve the same result, i.e.,a cytoplasmic TesA.

Overexpressed PldC(*TesA) results in hydrolysis of acyl groups fromendogenous acyl-ACP and acyl-CoA molecules. Cells expressing PldC(*TesA)must channel additional cellular carbon and energy to maintainproduction of acyl-ACP and acyl-coA molecules, which are required formembrane lipid synthesis. Thus, PldC(*TesA) expression results in a netincrease in total cellular lipid content. For instance, PldC(*TesA)expressed alone in Synechococcus doubles the total lipid content from10% of biomass to 20% of biomass, a result that can be further increasedby combining *TesA or related molecules with an introduced ACP and/or anintroduced Aas. Hence, certain embodiments employ an exogenous oroverexpressed cytoplasmic TesA (such as *TesA) in combination with anexogenous or overexpressed ACP, an exogenous or overexpressed Aas, orboth.

In certain embodiments, a thioesterase (TES) is an acyl-ACP thioesteraseand/or an acyl-CoA thioesterase. In particular embodiments, the TES is aTesA or TesB polypeptide from E. coli, or a cytoplasmic TesA variant(*TesA) variant having the sequence set forth in SEQ ID NO:121, or afragment or variant thereof.

Certain thioesterases have thioesterase activity only, i.e., they havelittle or no lysophospholipase activity. Examples of these thioesterasesinclude enzymes of the FatB family. FatB encoded enzymes typicallyhydrolyze saturated C14-C18 ACPs, preferentially 16:0 ACP, but they canalso hydrolyze 18:1 ACP. The production of medium chain (C8-C12) fattyacids in plants or seeds such as those of Cuphea spp. often results ofFatB enzymes that have chain length specificities for medium chain fattyacyl-ACPs. These medium chain FatB thioesterases are present in manyspecies with medium-chain fatty acids in their oil, including, forexample, California bay laurel, coconut, and elm, among others. Hence,FatB sequences may be derived from these and other organisms. Particularexamples include plant FatB acyl-ACP thioesterases such as C8, C12, C14,and C16 FatB thioesterases. Hence, in certain embodiments, the TES Is aFatB polypeptide, such as a C8, C12, C14, C16, or C18 FatB.

Specific examples of FatB thioesterases include the Cuphea hookerianaC8/C10 FatB thioesterase, the Umbellularia californica C12 FatB1thioesterase, the Cinnamomum camphora C14 FatB1 thioesterase, and theCuphea hookeriana C16 FatB1 thioesterase. In specific embodiments, thethioesterase Is a Cuphea hookeriana C8/C10 FatB, comprising the aminoacid sequence of SEQ ID NO:108 (full-length protein) or SEQ ID NO:109(mature protein without signal sequence). In particular embodiments, thethioesterase is a Umbellularia californica C12 FatB1, comprising theamino acid sequence of SEQ ID NO:110 (full-length protein) or SEQ IDNO:111 (mature protein without signal sequence). In certain embodiments,the thioesterase is a Cinnamomum camphora C14 FatB1, comprising theamino acid sequence of SEQ ID NO:112 (full-length protein) or SEQ IDNO:113 (mature protein without signal sequence). In particularembodiments, the thioesterase is a Cuphea hookeriana C16 FatB1,comprising the amino acid sequence of SEQ ID NO:114 (full-lengthprotein) or SEQ ID NO:115 (mature protein without signal sequence).

Acetyl Coenzyme A Carboxylases (ACCase).

Embodiments of the present disclosure optionally include one or moreexogenous (e.g., recombinantly introduced) or overexpressed ACCaseproteins. As used herein, an “acetyl CoA carboxylase” gene includes anypolynucleotide sequence encoding amino acids, such as protein,polypeptide or peptide, obtainable from any cell source, whichdemonstrates the ability to catalyze the carboxylation of acetyl-CoA toproduce malonyl-CoA under enzyme reactive conditions, and furtherincludes any naturally-occurring or non-naturally occurring variants ofan acetyl-CoA carboxylase sequence having such ability.

Acetyl-CoA carboxylase (ACCase) is a biotin-dependent enzyme thatcatalyses the irreversible carboxylation of acetyl-CoA to producemalonyl-CoA through its two catalytic activities, biotin carboxylase(BC) and carboxyltransferase (CT). The biotin carboxylase (BC) domaincatalyzes the first step of the reaction: the carboxylation of thebiotin prosthetic group that is covalently linked to the biotin carboxylcarrier protein (BCCP) domain. In the second step of the reaction, thecarboxyltransferase (CT) domain catalyzes the transfer of the carboxylgroup from (carboxy) biotin to acetyl-CoA. Formation of malonyl-CoA byacetyl-CoA carboxylase (ACCase) represents the commitment step for fattyacid synthesis, because malonyl-CoA has no metabolic role other thanserving as a precursor to fatty acids. Because of this reason,acetyl-CoA carboxylase represents a pivotal enzyme in the synthesis offatty acids.

In most prokaryotes, ACCase is a multi-subunit enzyme, whereas in mosteukaryotes it is a large, multi-domain enzyme. In yeast, the crystalstructure of the CT domain of yeast ACCase has been determined at 2.7Aresolution (Zhang et al., Science, 299:2064-2067 (2003). This structurecontains two domains, which share the same backbone fold. This foldbelongs to the crotonase/ClpP family of proteins, with a b-b-asuperhelix. The CT domain contains many insertions on its surface, whichare important for the dimerization of ACCase. The active site of theenzyme is located at the dimer interface.

Although Cyanobacteria, such as Synechococcus, express a native ACCaseenzyme, these bacteria typically do not produce or accumulatesignificant amounts of fatty acids. For example, Synechococcus in thewild accumulates fatty acids in the form of lipid membranes to a totalof about 4% by dry weight.

Given the role of ACCase in the commitment step of fatty acidbiosynthesis, embodiments of the present disclosure include methods ofincreasing the production of fatty acid biosynthesis, and, thus, lipidproduction, in Cyanobacteria by introducing one or more polynucleotidesthat encode an ACCase enzyme that is exogenous to the Cyanobacterium'snative genome. Embodiments of the present disclosure also include amodified Cyanobacterium, and compositions comprising the Cyanobacterium,comprising one or more polynucleotides that encode an ACCase enzyme thatis exogenous to the Cyanobacterium's native genome.

A polynucleotide encoding an ACCase enzyme may be isolated or obtainedfrom any organism, such as any prokaryotic or eukaryotic organism thatcontains an endogenous ACCase gene. Examples of eukaryotic organismshaving an ACCase gene are well-known in the art, and include variousanimals (e.g., mammals, fruit flies, nematodes), plants, parasites, andfungi (e.g., yeast such as S. cerevisiae and Schizosaccharomyces pombe).In certain embodiments, the ACCase encoding polynucleotide sequences areobtained from Synechococcus sp. PCC7002.

Examples of prokaryotic organisms that may be utilized to obtain apolynucleotide encoding an enzyme having ACCase activity include, butare not limited to, Escherichia coli, Legionella pneumophila, Listeriamonocytogenes, Streptococcus pneumoniae, Bacillus subtilis, Ruminococcusobeum ATCC 29174, marine gamma proteobacterium HTCC2080, Roseovarius sp.HTCC2601, Oceanicola granulosus HTCC2516, Bacteroides caccae ATCC 43185,Vibrio alginolyticus 12G01, Pseudoalteromonas tunicata D2, Marinobactersp. ELB17, marine gamma proteobacterium HTCC2143, Roseobacter sp.SK209-2-6, Oceanicola batsensis HTCC2597, Rhizobium leguminosarum bv.trifolii WSM1325, Nitrobacter sp. Nb-311A, Chloroflexus aggregans DSM9485, Chlorobaculum parvum, Chloroherpeton thalassium, Acinetobacterbaumannii, Geobacillus, and Stenotrophomonas maltophilla, among others.

Particular exemplary acetyl-CoA carboxylases (ACCase) comprise orconsist of a polypeptide sequence set forth in any of SEQ ID NOs:55, 45,46, 47, 48 or 49, or a fragment or variant thereof. SEQ ID NO:55 is thesequence of Saccharomyces cerevisiae acetyl-CoA carboxylase (yAcc1); SEQID NO:45 is Synechococcus sp. PCC 7002 AccA; SEQ ID NO:46 isSynechococcus sp. PCC 7002 AccB; SEQ ID NO:47 is Synechococcus sp. PCC7002 AccC; and SEQ ID NO:48 is Synechococcus sp. PCC 7002 AccD; and SEQID NO:49 is a Triticum aestivum ACCase. In certain embodiments, theintroduced ACCase is not native to the genome of the modifiedphotosynthetic microorganism.

Phosphatidic Acid Phosphatases (PAP).

As used herein, a “phosphatidate phosphatase” or “phosphatidic acidphosphatase” gene includes any polynucleotide sequence encoding aminoacids, such as protein, polypeptide or peptide, obtainable from any cellsource, which demonstrates the ability to catalyze the dephosphorylationof phosphatidate (PtdOH) under enzyme reactive conditions, yieldingdiacylglycerol (DAG) and inorganic phosphate, and further includes anynaturally-occurring or non-naturally occurring variants of aphosphatidate phosphatase sequence having such ability.

Phosphatidate phosphatases (PAP, 3-sn-phosphatidate phosphohydrolase)catalyze the dephosphorylation of phosphatidate (PtdOH), yieldingdiacylglycerol (DAG) and inorganic phosphate. This enzyme belongs to thefamily of hydrolases, specifically those acting on phosphoric monoesterbonds. The systematic name of this enzyme class is 3-sn-phosphatidatephosphohydrolase. Other names in common use include phosphatic acidphosphatase, acid phosphatidyl phosphatase, and phosphatic acidphosphohydrolase. This enzyme participates in at least 4 metabolicpathways: glycerolipid metabolism, glycerophospholipid metabolism, etherlipid metabolism, and sphingolipid metabolism.

PAP enzymes have roles in both the synthesis of phospholipids andtriacylglycerol through its product diacylglycerol, as well as thegeneration or degradation of lipid-signaling molecules in eukaryoticcells. PAP enzymes are typically classified as either Mg²⁺-dependent(referred to as PAP1 enzymes) or Mg²⁺-independent (PAP2 or lipidphosphate phosphatase (LPP) enzymes) with respect to their cofactorrequirement for catalytic activity. In both yeast and mammalian systems,PAP2 enzymes are known to be involved in lipid signaling. By contrast,PAP1 enzymes, such as those found in Saccharomyces cerevisiae, play arole in de novo lipid synthesis (Han, et al. J Biol Chem. 281:9210-9218,2006), thereby revealing that the two types of PAP are responsible fordifferent physiological functions.

In both yeast and higher eukaryotic cells, the PAP reaction is thecommitted step in the synthesis of the storage lipid triacylglycerol(TAG), which is formed from PtdOH through the intermediate DAG. Thereaction product DAG is also used in the synthesis of the membranephospholipids phosphatidylcholine (PtdCho) and phosphatidylethanolamine.The substrate PtdOH is used for the synthesis of all membranephospholipids (and the derivative inositol-containing sphingolipids)through the intermediate CDP-DAG. Thus, regulation of PAP activity mightgovern whether cells make storage lipids and phospholipids through DAGor phospholipids through CDP-DAG. In addition, PAP is involved in thetranscriptional regulation of phospholipid synthesis.

PAP1 enzymes have been purified and characterized from the membrane andcytosolic fractions of yeast, including a gene (Pah1, formerly known asSmp2) been identified to encode a PAP1 enzyme in S. cerevisiae. ThePah1-encoded PAP1 enzyme is found in the cytosolic and membranefractions of the cell, and its association with the membrane isperipheral in nature. As expected from the multiple forms of PAP1 thathave been purified from yeast, pah1Δ mutants still contain PAP1activity, indicating the presence of an additional gene or genesencoding enzymes having PAP1 activity.

Analysis of mutants lacking the Pah1-encoded PAP1 has provided evidencethat this enzyme generates the DAG used for lipid synthesis. Cellscontaining the pah1Δ mutation accumulate PtdOH and have reduced amountsof DAG and its acylated derivative TAG. Phospholipid synthesispredominates over the synthesis of TAG in exponentially growing yeast,whereas TAG synthesis predominates over the synthesis of phospholipidsin the stationary phase of growth. The effects of the pah1Δ mutation onTAG content are most evident in the stationary phase. For example,stationary phase cells devoid of the Pah1 gene show a reduction of >90%in TAG content. Likewise, the pah1Δ mutation shows the most markedeffects on phospholipid composition (e.g. the consequent reduction inPtdCho content) In the exponential phase of growth. The Importance ofthe Pah1-encoded PAP1 enzyme to cell physiology is further emphasizedbecause of its role in the transcriptional regulation of phospholipidsynthesis.

The requirement of Mg²⁺ ions as a cofactor for PAP enzymes is correlatedwith the catalytic motifs that govern the phosphatase reactions of theseenzymes. For example, the Pah1-encoded PAP1 enzyme has a DxDxT (SEQ IDNO:198) catalytic motif within a haloacid dehalogenase (HAD)-like domain(“x” is any amino acid). This motif is found in a superfamily ofMg²⁺-dependent phosphatase enzymes, and its first aspartate residue isresponsible for binding the phosphate moiety in the phosphatasereaction. By contrast, the DPP1- and LPP1-encoded PAP2 enzymes contain athree-domain lipid phosphatase motif that is localized to thehydrophilic surface of the membrane. This catalytic motif, whichcomprises the consensus sequences KxxxxxxRP (domain 1) (SEQ ID NO:116),PSGH (domain 2) (SEQ ID NO:117), and SRxxxxxHxxxD (domain 3) (SEQ IDNO:118), is shared by a superfamily of lipid phosphatases that do notrequire Mg²⁺ ions for activity. The conserved arginine residue in domain1 and the conserved histidine residues in domains 2 and 3 may beessential for the catalytic activity of PAP2 enzymes. Accordingly, aphosphatidate phosphatase polypeptide may comprise one or more of theabove-described catalytic motifs.

A polypeptide having a phosphatidate phosphatase enzymatic activity maybe obtained from any organism having a suitable, endogenousphosphatidate phosphatase gene. Examples of organisms that may be usedto obtain a phosphatidate phosphatase encoding polynucleotide sequenceinclude, but are not limited to, Homo sapiens, Mus musculus, Rattusnorvegicus, Bos taurus, Drosophila melanogaster, Arabidopsis thaliana,Magnaporthe grisea, Saccharomyces cerevisiae, Schizosaccharomyces pombe,Cryptococcus neoformans, and Bacillus pumilus, among others. Specificexamples of PAP enzymes include Pah1 from S. cerevisiae, PgpB from E.coli, and PAP from PCC6803.

In certain embodiments, a phosphatidate phosphatase polypeptidecomprises or consists of a polypeptide sequence set forth in SEQ IDNO:131, or a fragment or variant thereof. SEQ ID NO:131 is the sequenceof Saccharomyces cerevisiae phosphatidate phosphatase (yPah1). Incertain embodiments, the polypeptide sequence of the PAP is encoded bythe E. coli PgpB gene, and/or the PAP gene from Synechocystis sp.PCC6803.

Triacylglycerol (TAG) Hydrolases.

Certain embodiments relate to the use of exogenous or overexpressed TAGhydrolases (or TAG lipases) to increase production of TAGs in aTAG-producing strain. For instance, certain embodiments may utilize aTAG hydrolase in combination with a DGAT, and optionally a TES. Theseembodiments may then further utilize an ACP, an Aas, or both, any of thelipid biosynthesis proteins described herein, and/or any of themodifications to glycogen production and storage described herein.Hence, as noted above, TAG hydrolases may be used in TAG-producingstrains (e.g., DGAT-expressing strains) with or without an ACP or Aas.

TAG hydrolases are carboxylesterases that are typically specific forinsoluble long chain fatty acid TAGs. Carboxylesterases catalyze thechemical reaction:carboxylic ester+H₂O

alcohol+carboxylate

Thus, the two substrates of this enzyme are carboxylic ester and H₂O,whereas its two products are alcohol and carboxylate. According to onenon-limiting theory, it is understood that TAG hydrolase expression (oroverexpression) in a TAG producing strain (e.g., DGAT/ACP, DGAT/Aas,DGAT/ACP/Aas) releases acyl chains to not only increase accumulation offree fatty acids (FFA), but also increase the amount of free 1, 2diacylglycerol (DAG). This free DAG then serves as a substrate for DGAT,and thereby allows increased TAG production, especially in the presenceof over-expressed ACP, Aas, or both. Accordingly, certain embodimentsemploying a TAG hydrolase produce increased amounts of TAG, relative,for example, to a DGAT only-expressing microorganism. In certainembodiments, the TAG hydrolase is specific for TAG and not DAG, i.e., itpreferentially acts on TAG relative to DAG.

Non-limiting examples of TAG hydrolases include SDP1 (SUGAR-DEPENDENT1)triacylglycerol lipase from Arobidopsis thaliana (SEQ ID NO:170),ACIAD1335 from Acinetobacter sp. ADP1 (SEQ ID NO:171), TG14P from S.cerevisiae (SEQ ID NO:172), and RHA1_ro04722 (YP_704665) TAG lipase fromRhodococcus (SEQ ID NO:173). Additional putative lipases/esterases fromRhodococcus include RHA1_ro01602 lipase/esterase (see SEQ ID NOs:156 and174 for polynucleotide and polypeptide sequence, respectively), andRHA1_ro06856 lipase/esterase (see SEQ ID NOs:119 and 120 forpolynucleotide and polypeptide sequence, respectively).

Fatty Acyl-COA Synthetases.

Certain embodiments relate to the use of overexpressed fatty acyl-CoAsynthetases to increase activation of fatty acids, and thereby increaseproduction of TAGs in a TAG-producing strain (e.g., a DGAT-expressingstrain). For instance, certain embodiments may utilize an acyl-ACPreductase in combination with a fatty acyl-CoA synthetase and a DGAT.These embodiments may then further utilize an ACP, an ACCase, or both,and/or any of the modifications to glycogen production and storage orglycogen breakdown described herein.

Fatty acyl-CoA synthetases activate fatty acids for metabolism bycatalyzing the formation of fatty acyl-CoA thioesters. Fatty acyl-CoAthioesters can then serve not only as substrates for beta-oxidation, atleast in bacteria capable of growing on fatty acids as a sole source ofcarbon (e.g., E. coli, Salmonella), but also as acyl donors inphospholipid biosynthesis. Many fatty acyl-CoA synthetases arecharacterized by two highly conserved sequence elements, an ATP/AMPbinding motif, which is common to enzymes that form an adenylatedintermediate, and a fatty acid binding motif.

According to one non-limiting theory, certain embodiments may employfatty acyl-CoA synthetases to increase activation of free fatty acids,which can then be incorporated into TAGs, mainly by the DGAT-expressing(and thus TAG-producing) photosynthetic microorganisms described herein.Hence, fatty acyl-CoA synthetases can be used in any of the embodimentsdescribed herein, such as those that produce increased levels of freefatty acids, where it is desirable to turn free fatty acids into TAGs.As noted above, these free fatty acids can then be activated by fattyacyl-CoA synthetases to generate acyl-CoA thioesters, which can thenserve as substrates by DGAT to produce increased levels of TAGs.

One exemplary fatty acyl-CoA synthetase includes the FadD gene from E.coli (SEQ ID NOS:16 and 17 for nucleotide and polypeptide sequence,respectively), which encodes a fatty acyl-CoA synthetase havingsubstrate specificity for medium and long chain fatty acids. Otherexemplary fatty acyl-CoA synthetases include those derived from S.cerevisiae; Faa1p can use C12-C16 acyl-chains in vitro (see SEQ IDNOS:18 and 19 for nucleotide and polypeptide sequence, respectively),Faa2p shows a less restricted specificity ranging from C7-C17 (see SEQID NOS:20 and 21 for nucleotide and polypeptide sequence, respectively),and Faa3p, together with that of DGAT1, enhances lipid accumulation inthe presence of exogenous fatty acids in S. cerevisiae (see SEQ ID NO:22and 23 for nucleotide and polypeptide sequence, respectively). SEQ IDNO:22 is codon-optimized for expression in S. elongatus PCC7942.

Lipases/Phospholipases.

In various embodiments, modified photosynthetic microorganisms, e.g.,Cyanobacteria, of the present disclosure further comprise one or moreexogenous or introduced nucleic acids that encode a polypeptide having alipase or phospholipase activity, or a fragment or variant thereof.Lipases, including phospholipases, lysophospholipases, thioesterases,and enzymes having one, two, or all three of these activities, typicallycatalyze the hydrolysis of ester chemical bonds in lipid substrates.Without wishing to be bound by any one theory, in certain exemplaryembodiments the expression of one or more phospholipases can generatefatty acids from membrane lipids, which may then be used by the ACPand/or Aas to make acyl-ACPs. These acyl-ACPs, for example, can thenfeed into the triglyceride synthesis pathways, thereby increasingtriglyceride (TAG) production.

A phospholipase is an enzyme that hydrolyzes phospholipids into fattyacids and other lipophilic substances. There are four major classes,termed A, B, C and D distinguished by what type of reaction theycatalyze. Phospholipase A1 cleaves the SN-1 acyl chain, whilePhospholipase A2 cleaves the SN-2 acyl chain, releasing arachidonicacid. Phospholipase B cleaves both SN-1 and SN-2 acyl chains, and isalso known as a lysophospholipase. Phospholipase C cleaves before thephosphate, releasing diacylglycerol and a phosphate-containing headgroup. Phospholipases C play a central role in signal transduction,releasing the second messenger, inositol triphosphate. Phospholipase Dcleaves after the phosphate, releasing phosphatidic acid and an alcohol.Types C and D are considered phosphodiesterases. In various embodiments,one or more phospholipase from any one of these classes may be used,alone or in any combination.

As noted above, phospholipases (PLA1,2) act on phospholipids ofdifferent kinds including phosphatidyl glycerol, the major phospholipidin Cyanobacteria, by cleaving the acyl chains off the sn1 or sn2positions (carbon 1 or 2 on the glycerol backbone); some are selectivefor sn1 or sn2, others act on both. Lysophospholipases act onlysophospholipids, which can be the product of phospholipases or onlysophosphatidic acid, a normal intermediate of the de novo phosphatidicacid synthesis pathway, e.g., 1-acyl-DAG-3-phosphate.

Merely by way of non-limiting theory, it is understood that in certainembodiments, phospholipases and/or lysophospholipases can cleave offacyl chains from phospholipids or lysophospholipids and thus deregulatethe normal recycling of the lipid membranes, including both cellmembrane and thylakoid membranes, which then leads to accumulation offree fatty acids (FFAs). In certain embodiments (e.g., TesA strains),these FFAs may accumulate extracellularly. In other embodiments (e.g.,ACP and/or Aas over-expressing microorganisms), FFAs can be convertedinto acyl-ACPs by acyl ACP synthase (Aas) in a strain that alsoover-expresses ACP. In certain embodiments (e.g., DGAT-containingmicroorganisms), these acyl-ACPs can then serve as substrates for DGATto make TAGs.

In other embodiments, phospholipases can be over-expressed to generatelyshophospholipids and acyl chains. The lysophospholipids can then serveas substrates for a lysophospholipase, which cleaves off the remainingacyl chain. In some embodiments, these acyl chains can either accumulateas FFAs, or in other embodiments may serve as substrates of Acyl ACPsynthase (Aas) to generate acyl-ACPs, which can then be used by DGAT tomake TAGs.

Particular examples of phospholipase C enzymes include those derivedfrom eukaryotes such as mammals and parasites, in addition to thosederived from bacteria. Examples include phosphoinositide phospholipase C(EC 3.1.4.11), the main form found in eukaryotes, especially mammals,the zinc-dependent phospholipase C family of bacterial enzymes (EC3.1.4.3) that includes alpha toxins, phosphatidylinositoldiacylglycerol-lyase (EC 4.6.1.13), a related bacterial enzyme, andglycosylphosphatidylinositol diacylglycerol-lyase (EC 4.6.1.14), atrypanosomal enzyme.

In particular embodiments, the present disclosure contemplates using alysophospholipase. A lysophospholipase is an enzyme that catalyzes thechemical reaction:2-lysophosphatidic acid+H₂O

glycerol-3-phosphate+a carboxylate

Thus, the two substrates of this enzyme are 2-lysophosphatidylcholineand H₂O, whereas its two products are glycerophosphocholine andcarboxylate.

Lysophospholipase are members of the hydrolase family, specificallythose acting on carboxylic ester bonds. Lysophospholipases participatein glycerophospholipid metabolism. Examples of lysophospholipasesinclude, but are not limited to, 2-Lysophosphatidylcholineacylhydrolase, Lecithinase B, Lysolecithinase, Phospholipase B,Lysophosphatidase, Lecitholipase, Phosphatidase B,Lysophosphatidylcholine hydrolase, Lysophospholipase A1,Lysophospholipase L1 (TesA), Lysophopholipase L2, TesB,Lysophospholipase transacylase, Neuropathy target esterase, NTE,NTE-LysoPLA, NTE-lysophospholipase, and Vu Patatin 1 protein. Inparticular embodiments, lysophospholipases utilized according to thepresent disclosure are derived from a bacteria, e.g., E. coli, or aplant. Any of these lysophospholipases may be used according to variousembodiments of the present Invention.

Certain lysophospholipases, such as Lysophospholipase L1 (also referredto as PldC or TesA) are periplasmically-localized orcytoplasmically-localized enzymes that have both lysophospholipase andthioesterase activity, as described above. Hence, certain thioesterasessuch as TesA can also be characterized as lysophospholipases. A mutantlysophospholipase described herein, PldC(*TesA), is not exported to theperiplasm due to deletion of an N-terminal amino acid sequence requiredfor proper transport of TesA from the cytoplasm to the periplasm. Thisresults in a cytoplasmic-localized PldC(*TesA) protein that has accessto endogenous acyl-ACP and acyl-CoA intermediates. OverexpressedPldC(*TesA) results in hydrolysis of acyl groups from endogenousacyl-ACP and acyl-CoA molecules. Cells expressing PldC(*TesA) mustchannel additional cellular carbon and energy to maintain production ofacyl-ACP and acyl-coA molecules, which are required for membrane lipidsynthesis. Thus, PldC(*TesA) expression results in a net Increase incellular lipid content. As described herein, PldC(*TesA) is expressed inSynechococcus lipid content doubles from 10% of biomass to 20% ofbiomass.

In certain embodiments, lysophospholipases utilized according to thepresent disclosure have both phospholipase and thioesterase activities.Examples of lysophospholipases that have both activities include, e.g.,Lysophospholipase L1 (TesA), such as E. coli Lysophospholipase L1, aswell as fragments and variants thereof, including those described in theparagraph above. As a phospholipase, certain embodiments may employ TesAvariants having only lysophospholipase activity, including variants withreduced or no thioesterase activity.

In particular embodiments, the phospholipase is a bacterialphospholipase, e.g., lysophospholipase, or a fragment or variantthereof, e.g., a phospholipase derived from Escherichia coli, S.cerevisiae, Rhodococcus, Streptomyces or Acinetobacter species.

Additional non-limiting examples of phospholipases include phospholipaseA1 (PldA) from Acinetobacter sp. ADP1, phospholipase A (PldA) from E.coli, phospholipase from Streptomyces coelicolor A3(2), phospholipase A2(PLA2-α) from Arobidopsis thaliana; phospholipase A1/triacylglycerollipase (DAD1; Defective Anther Dehiscence 1) from Arobidopsis thaliana,chloroplast DONGLE from Arobidopsis thaliana, patatin-like protein fromArabidopsis thaliana, and patatin from Anabaena variabilis ATCC 29413.Additional non-limiting examples of lysophospholipases includephospholipase B (Plb1p) from Saccharomyces cerevisiae S288c,phospholipase B (Plb2p) from Saccharomyces cerevisiae S288c, ACIAD1057(tesA homolog) from Acinetobacter ADP1, ACIAD1943 lysophospholipase fromAcinetobacter ADP1, and a lysophospholipase (YP_702320; RHA1_ro02357)from Rhodococcus.

In particular embodiments, the encoded phospholipase comprises orconsists of a Lysophospholipase L1 (TesA), Lysophospholipase L2, TesB,or Vu patatin 1 protein, or a homolog, fragment, or variant thereof. Incertain embodiments, the Lysophospholipase L1 (TesA), LysophospholipaseL2, or TesB is a bacterial Lysophospholipase L1 (TesA),Lysophospholipase L2, or TesB, such as an E. coli Lysophospholipase L1(TesA) having the wild-type sequence set forth in SEQ ID NO:133, an E.coli Lysophospholipase L2 having the wild-type sequence set forth in SEQID NO:137, or an E. coli TesB having the wild-type sequence set forth inSEQ ID NO:134. In particular embodiment, the Vu patatin 1 protein hasthe wild-type sequence set forth in SEQ ID NO:138.

In particular embodiments, the phospholipase is modified such that itlocalizes predominantly to the cytoplasm instead of the periplasm. Forexample, the phospholipase may have a deletion or mutation in a regionassociated with periplasmic localization. In particular embodiments, thephospholipase variant is derived from Lysophospholipase L1 (TesA) orTesB. In certain embodiments, the Lysophospholipase L1 (TesA) or TesBvariant is a bacterial Lysophospholipase L1 (TesA) or TesB variant, suchas a cytoplasmic E. coli Lysophospholipase L1 (PldC(*TesA)) varianthaving the sequence set forth in SEQ ID NO:121.

Additional examples of phospholipase polypeptide sequences includephospholipase A1 (PldA) from Acinetobacter sp. ADP1 (SEQ ID NO:157),phospholipase A (PldA) from E. coli (SEQ ID NO:158), phospholipase fromStreptomyces coelicolor A3(2) (SEQ ID NO:159), phospholipase A2 (PLA2-α)from Arabidopsis thaliana (SEQ ID NO:160). phospholipaseA1/triacylglycerol lipase (DAD1; Defective Anther Dehiscence 1) fromArabidopsis thaliana (SEQ ID NO:161), chloroplast DONGLE fromArabidopsis thaliana (SEQ ID NO:162), patatin-like protein fromArabidopsis thaliana (SEQ ID NO:163), and patatin from Anabaenavariabilis ATCC 29413 (SEQ ID NO:164). Additional non-limiting examplesof lysophospholipase polypeptide sequences include phospholipase B(Plb1p) from Saccharomyces cerevisiae S288c (SEQ ID NO:165),phospholipase B (Plb2p) from Saccharomyces cerevisiae S288c (SEQ IDNO:166), ACIAD1057 (TesA homolog) from Acinetobacter ADP1 (SEQ IDNO:167), ACIAD1943 lysophospholipase from Acinetobacter ADP1 (SEQ IDNO:168), and a lysophospholipase (YP_702320; RHA1_ro02357) fromRhodococcus (SEQ ID NO:169).

Fatty Acyl Reductase.

Certain embodiments relate to the use of overexpressed fatty acylreductases to increase synthesis of fatty alcohols, and thereby increaseproduction of wax esters in a WE-producing strain (e.g., aDGAT-expressing strain). For instance, certain embodiments may utilize afatty acyl reductase, possibly in combination with an acyl-ACPreductase, and a DGAT. These embodiments may then further utilize anACP, an ACCase, or both, and/or any of the modifications to glycogenproduction and storage or glycogen breakdown described herein.

Fatty Acyl Reductases catalyze the two step reduction of acyl-ACP's oracyl-COA's to acyl alcohols, also known as fatty alcohols. The firststep proceeds via an acyl aldehyde intermediate, which is then convertedin a second step to a fatty alcohol. These same enzymes can alsodirectly reduce fatty aldehydes to fatty alcohols (i.e. step two only).In this case they are sometimes referred to as fatty aldehydereductases. Fatty alcohols can serve as a substrate for wax esterbiosynthesis by a DGAT. Many fatty acyl reductases are characterized bythree conserved sequence elements. There is an NADPH binding motif, amotif characteristic of the catalytic site of NADP-utilizing enzymes,and a conserved C-terminal domain, referred to as the Male Sterile 2domain, that is of unknown function (see Hofvander et al., FEBS Letters(2011) pp 3583-3543)

According to one non-limiting theory, certain embodiments may employfatty acyl reductases to increase synthesis of fatty alcohols, which canthen be incorporated into WE's, mainly by the DGAT-expressing (and thusWE-producing) photosynthetic microorganisms described herein. Hence,fatty acyl reductases can be used in any of the embodiments describedherein, such as those that produce increased levels of free fattyalcohols, where it is desirable to turn these into WE's. As noted above,these free fatty alcohols can then be esterified to fatty acids (in theform of acyl-ACP) by DGATs to generate WE's.

One exemplary fatty acyl reductase includes a gene from Marinobacteraquaeolei VT8, genbank accession number YP_959769.1 (see SEQ ID NOs:224and 225 for polypeptide and polynucleotide sequence, respectively).Others include a gene from Simmondsia chinesis (Jojoba) AF149917 (seeSEQ ID NOs:226 and 227 for polypeptide and polynucleotide sequence,respectively); a gene from Euglena gracilis GU733919 (see SEQ ID NOs:228and 229 for polypeptide and polynucleotide sequence, respectively); agene from Hahella chejuensis YP 436183 (see SEQ ID NOs:230 and 231 forpolypeptide and polynucleotide sequence, respectively); a gene fromPhotobacterium profundum SS59 YP 130411.1 (see SEQ ID NOs:232 and 233for polypeptide and polynucleotide sequence, respectively); a gene fromMarinobacter algicola DG893 ZP 01892457 (see SEQ ID NOs:234 and 235 forpolypeptide and polynucleotide sequence, respectively); a gene fromMarinobacter adhaerens HP15 ADP96574 (see SEQ ID NOs:236 and 237 forpolypeptide and polynucleotide sequence, respectively); a gene fromArabidopsis thaliana CERF NM 119537 (see SEQ ID NOs:238 and 239 forpolypeptide and polynucleotide sequence, respectively); a gene fromArabidopsis thaliana At3g56700 NC 003074 (see SEQ ID NOs:240 and 241 forpolypeptide and polynucleotide sequence, respectively); a gene fromAradopsis thaliana Atg22500 NC 003076 (see SEQ ID NOs:242 and 243 forpolypeptide and polynucleotide sequence, respectively); and a gene fromTriticum aestivum (Wheat bread) AJ459250 (see SEQ ID NOs:244 and 245 forpolypeptide and polynucleotide sequence, respectively).

(iii) Glycogen Synthesis, Storage, and Breakdown

In particular embodiments, a modified photosynthetic microorganismfurther comprises additional modifications, such that it has reducedexpression of one or more genes associated with a glycogen synthesis orstorage pathway and/or increased expression of one or morepolynucleotides that encode a protein associated with a glycogenbreakdown pathway, or a functional variant of fragment thereof.

In various embodiments, modified photosynthetic microorganisms, e.g.,Cyanobacteria, of the present disclosure have reduced expression of oneor more genes associated with glycogen synthesis and/or storage. Inparticular embodiments, these modified photosynthetic microorganismshave a mutated or deleted gene associated with glycogen synthesis and/orstorage. In particular embodiments, these modified photosyntheticmicroorganisms comprise a vector that includes a portion of a mutated ordeleted gene, e.g., a targeting vector used to generate a knockout orknockdown of one or more alleles of the mutated or deleted gene. Incertain embodiments, these modified photosynthetic microorganismscomprise an antisense RNA or siRNA that binds to an mRNA expressed by agene associated with glycogen synthesis and/or storage.

In certain embodiments, modified photosynthetic microorganisms, e.g.,Cyanobacteria, of the present disclosure comprise one or more exogenousor introduced nucleic acids that encode a polypeptide having an activityassociated with a glycogen breakdown or triglyceride or fatty acidbiosynthesis, including but not limited to any of those describedherein. In particular embodiments, the exogenous nucleic acid does notcomprise a nucleic acid sequence that is native to the microorganism'sgenome. In particular embodiments, the exogenous nucleic acid comprisesa nucleic acid sequence that is native to the microorganism's genome,but it has been introduced into the microorganism, e.g., in a vector orby molecular biology techniques, for example, to increase expression ofthe nucleic acid and/or its encoded polypeptide in the microorganism.

Glycogen Biosynthesis and Storage.

Glycogen is a polysaccharide of glucose, which functions as a means ofcarbon and energy storage in most cells, including animal and bacterialcells. More specifically, glycogen is a very large branched glucosehomopolymer containing about 90% α-1,4-glucosidic linkages and 10% α-1,6linkages. For bacteria in particular, the biosynthesis and storage ofglycogen in the form of α-1,4-polyglucans represents an importantstrategy to cope with transient starvation conditions in theenvironment.

Glycogen biosynthesis involves the action of several enzymes. Forinstance, bacterial glycogen biosynthesis occurs generally through thefollowing general steps: (1) formation of glucose-1-phosphate, catalyzedby phosphoglucomutase (Pgm), followed by (2) ADP-glucose synthesis fromATP and glucose 1-phosphate, catalyzed by glucose-1-phosphateadenylyltransferase (GlgC), followed by (3) transfer of the glucosylmoiety from ADP-glucose to a pre-existing α-1,4 glucan primer, catalyzedby glycogen synthase (GlgA). This latter step of glycogen synthesistypically occurs by utilizing ADP-glucose as the glucosyl donor forelongation of the α-1,4-glucosidic chain.

In bacteria, the main regulatory step in glycogen synthesis takes placeat the level of ADP-glucose synthesis, or step (2) above, the reactioncatalyzed by glucose-1-phosphate adenylyltransferase (GlgC), also knownas ADP-glucose pyrophosphorylase (see, e.g., Ballicora et al.,Microbiology and Molecular Biology Reviews 6:213-225, 2003). Incontrast, the main regulatory step in mammalian glycogen synthesisoccurs at the level of glycogen synthase. As shown herein, by alteringthe regulatory and/or other active components in the glycogen synthesispathway of photosynthetic microorganisms such as Cyanobacteria, andthereby reducing the biosynthesis and storage of glycogen, the carbonthat would have otherwise been stored as glycogen can be utilized by thephotosynthetic microorganism to synthesize other carbon-based storagemolecules, such as lipids, fatty acids, and triglycerides.

Therefore, certain modified photosynthetic microorganisms, e.g.,Cyanobacteria, of the present disclosure may comprise a mutation,deletion, or any other alteration that disrupts one or more of thesesteps (i.e., renders the one or more steps “non-functional” with respectto glycogen biosynthesis and/or storage), or alters any one or more ofthe enzymes directly involved in these steps, or the genes encodingthem. As noted above, such modified photosynthetic microorganisms, e.g.,Cyanobacteria, are typically capable of producing and/or accumulating anincreased amount of lipids, such as fatty acids, as compared to a wildtype photosynthetic microorganism. Certain exemplary glycogenbiosynthesis genes are described below.

Phosphoglucomutase Gene (Pgm).

In one embodiment, a modified photosynthetic microorganism, e.g., aCyanobacteria, expresses a reduced amount of the phosphoglucomutasegene. In particular embodiments, it may comprise a mutation or deletionin the phosphoglucomutase gene, including any of its regulatory elements(e.g., promoters, enhancers, transcription factors, positive or negativeregulatory proteins, etc.). Phosphoglucomutase (Pgm), encoded by thegene pgm, catalyzes the reversible transformation of glucose 1-phosphateinto glucose 6-phosphate, typically via the enzyme-bound intermediate,glucose 1,6-biphosphate (see, e.g., Lu et al., Journal of Bacteriology176:5847-5851, 1994). Although this reaction is reversible, theformation of glucose-6-phosphate is markedly favored.

However, typically when a large amount of glucose-6-phosphate ispresent, Pgm catalyzes the phosphorylation of the 1-carbon and thedephosphorylation of the c-carbon, resulting in glucose-1-phosphate. Theresulting glucose-1-phosphate is then converted to UDP-glucose by anumber of intermediate steps, including the catalytic activity of GlgC,which can then be added to a glycogen storage molecule by the activityof glycogen synthase, described below. Thus, under certain conditions,the Pgm enzyme plays an intermediary role in the biosynthesis andstorage of glycogen.

The pgm gene is expressed in a wide variety of organisms, includingmost, if not all, Cyanobacteria. The pgm gene is also fairly conservedamong Cyanobacteria, as can be appreciated upon comparison of SEQ IDNOs:24 (S. elongatus PCC7942), 25 (Synechocystis sp. PCC6803), and 26(Synechococcus sp. WH8102), 79 (Synechococcus RCC307), and 80(Synechococcus 7002), which provide the polynucleotide sequences ofvarious pgm genes from Cyanobacteria.

Deletion of the pgm gene in Cyanobacteria, such as Synechococcus, hasbeen demonstrated herein for the first time to reduce the accumulationof glycogen in the Cyanobacteria, and also to increase the production ofother carbon-based products, such as lipids and fatty acids.

Glucose-1-Phosphate Adenylyltransferase (glgC).

In one embodiment, a modified photosynthetic microorganism, e.g., aCyanobacteria, expresses a reduced amount of a glucose-1-phosphateadenylyltransferase (glgC) gene. In certain embodiments, it may comprisea mutation or deletion in the glgC gene, including any of its regulatoryelements. The enzyme encoded by the glgC gene (e.g., EC 2.7.7.27)participates generally in starch, glycogen and sucrose metabolism bycatalyzing the following chemical reaction:ATP+alpha-D-glucose1-phosphate

diphosphate+ADP-glucose

Thus, the two substrates of this enzyme are ATP and alpha-D-glucose1-phosphate, whereas its two products are diphosphate and ADP-glucose.The glgC-encoded enzyme catalyzes the first committed and rate-limitingstep in starch biosynthesis in plants and glycogen biosynthesis inbacteria. It is the enzymatic site for regulation of storagepolysaccharide accumulation in plants and bacteria, being allostericallyactivated or inhibited by metabolites of energy flux.

The enzyme encoded by the glgC gene belongs to a family of transferases,specifically those transferases that transfer phosphorus-containingnucleotide groups (i.e., nucleotidyl-transferases). The systematic nameof this enzyme class is typically referred to asATP:alpha-D-glucose-1-phosphate adenylyltransferase. Other names incommon use include ADP glucose pyrophosphorylase, glucose 1-phosphateadenylyltransferase, adenosine diphosphate glucose pyrophosphorylase,adenosine diphosphoglucose pyrophosphorylase, ADP-glucosepyrophosphorylase, ADP-glucose synthase, ADP-glucose synthetase, ADPGpyrophosphorylase, and ADP:alpha-D-glucose-1-phosphateadenylyltransferase.

The glgC gene is expressed in a wide variety of plants and bacteria,including most, if not all, Cyanobacteria. The glgC gene is also fairlyconserved among Cyanobacteria, as can be appreciated upon comparison ofSEQ ID NOs:27 (S. elongatus PCC7942), 28 (Synechocystis sp. PCC6803), 29(Synechococcus sp. PCC 7002), 30 (Synechococcus sp. WH8102), 31(Synechococcus sp. RCC 307), 32 (Trichodesmium erythraeum IMS 101), 33(Anabaena varibilis), and 34 (Nostoc sp. PCC 7120), which describe thepolynucleotide sequences of various glgC genes from Cyanobacteria.

Deletion of the glgC gene in Cyanobacteria, such as Synechococcus,reduces the accumulation of glycogen in the Cyanobacteria, and increasesthe production of other carbon-based products, such as lipids and fattyacids.

Glycogen Synthase (glgA).

In one embodiment, a modified photosynthetic microorganism, e.g., aCyanobacteria, expresses a reduced amount of a glycogen synthase gene.In particular embodiments, it may comprise a deletion or mutation in theglycogen synthase gene, including any of is regulatory elements.Glycogen synthase (GlgA), also known as UDP-glucose-glycogenglucosyltransferase, is a glycosyltransferase enzyme that catalyses thereaction of UDP-glucose and (1,4-α-D-glucosyl)_(n) to yield UDP and(1,4-α-D-glucosyl)_(n+1). Glycogen synthase is an α-retainingglucosyltransferase that uses ADP-glucose to incorporate additionalglucose monomers onto the growing glycogen polymer. Essentially, GlgAcatalyzes the final step of converting excess glucose residues one byone into a polymeric chain for storage as glycogen.

Classically, glycogen synthases, or α-1,4-glucan synthases, have beendivided into two families, animal/fungal glycogen synthases andbacterial/plant starch synthases, according to differences in sequence,sugar donor specificity and regulatory mechanisms. However, detailedsequence analysis, predicted secondary structure comparisons, andthreading analysis show that these two families are structurally relatedand that some domains of animal/fungal synthases were acquired to meetthe particular regulatory requirements of those cell types.

Crystal structures have been established for certain bacterial glycogensynthases (see, e.g., Buschiazzo et al., The EMBO Journal 23, 3196-3205,2004). These structures show that reported glycogen synthase folds intotwo Rossmann-fold domains organized as in glycogen phosphorylase andother glycosyltransferases of the glycosyltransferases superfamily, witha deep fissure between both domains that includes the catalytic center.The core of the N-terminal domain of this glycogen synthase consists ofa nine-stranded, predominantly parallel, central β-sheet flanked on bothsides by seven α-helices. The C-terminal domain (residues 271-456) showsa similar fold with a six-stranded parallel β-sheet and nine α-helices.The last α-helix of this domain undergoes a kink at position 457-460,with the final 17 residues of the protein (461-477) crossing over to theN-terminal domain and continuing as α-helix, a typical feature ofglycosyltransferase enzymes.

These structures also show that the overall fold and the active sitearchitecture of glycogen synthase are remarkably similar to those ofglycogen phosphorylase, the latter playing a central role in themobilization of carbohydrate reserves, indicating a common catalyticmechanism and comparable substrate-binding properties. In contrast toglycogen phosphorylase, however, glycogen synthase has a much widercatalytic cleft, which is predicted to undergo an important interdomain‘closure’ movement during the catalytic cycle.

Crystal structures have been established for certain GlgA enzymes (see,e.g., Jin et al., EMBO J 24:694-704, 2005, incorporated by reference).These studies show that the N-terminal catalytic domain of GlgAresembles a dinucleotide-binding Rossmann fold and the C-terminal domainadopts a left-handed parallel beta helix that is involved in cooperativeallosteric regulation and a unique oligomerization. Also, communicationbetween the regulator-binding sites and the active site involves severaldistinct regions of the enzyme, including the N-terminus, theglucose-1-phosphate-binding site, and the ATP-binding site.

The glgA gene is expressed in a wide variety of cells, including animal,plant, fungal, and bacterial cells, including most, if not all,Cyanobacteria. The glgA gene is also fairly conserved amongCyanobacteria, as can be appreciated upon comparison of SEQ ID NOs:35(S. elongatus PCC7942), 36 (Synechocystis sp. PCC6803), 37(Synechococcus sp. PCC 7002), 38 (Synechococcus sp. WH8102), 39(Synechococcus sp. RCC 307), 40 (Trichodesmium erythraeum IMS 101), 41(Anabaena variabilis), and 42 (Nostoc sp. PCC 7120), which describe thepolynucleotide sequences of various glgA genes from Cyanobacteria.

Glycogen Breakdown.

In certain embodiments, a modified photosynthetic microorganism of thepresent disclosure expresses an increased amount of one or morepolypeptides associated with a glycogen breakdown pathway. In particularembodiments, the one or more polypeptides include glycogen phosphorylase(GlgP), glycogen isoamylase (GlgX), glucanotransferase (MalQ),phosphoglucomutase (Pgm), glucokinase (Glk), and/or phosphoglucoseisomerase (Pgi), or a functional fragment or variant thereof, including,for example, those provided in SEQ ID NOs:68, 70, 72, 73, 83 or 85.Examples of additional Pgm polypeptide sequences useful according to thepresent disclosure are provided in SEQ ID NOs:74, 76, 77, 79, and 81.Pgm, Glk, and Pgi are bidirectional enzymes that can promote glycogensynthesis or breakdown depending on conditions.

(iv) Polypeptide Variants and Fragments

As noted above, embodiments of the present disclosure include variantsand fragments of any of the reference polypeptides and polynucleotidesdescribed herein (see, e.g., the Sequence Listing). Variant polypeptidesare biologically active, that is, they continue to possess the enzymaticactivity of a reference polypeptide. Such variants may result from, forexample, genetic polymorphism and/or from human manipulation.

Biologically active variants of a reference polypeptide will have atleast 40%, 50%, 60%, 70%, generally at least 75%, 80%, 85%, usuallyabout 90% to 95% or more, and typically about 97% or 98% or moresequence similarity or sequence identity to the amino acid sequence fora reference protein as determined by sequence alignment programsdescribed elsewhere herein using default parameters. A biologicallyactive variant of a reference polypeptide may differ from that proteingenerally by as much 200, 100, 50 or 20 amino acid residues or suitablyby as few as 1-15 amino acid residues, as few as 1-10, such as 6-10amino acid residues, including about 20, 19, 18, 17, 16, 15, 14, 13, 12,11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or even 1 amino acid residues. In someembodiments, a variant polypeptide differs from the reference sequencesreferred to herein (see, e.g., the Sequence Listing) by at least one butby less than 15, 10 or 5 amino acid residues. In other embodiments, itdiffers from the reference sequences by at least one residue but lessthan 20%, 15%, 10% or 5% of the residues.

A biologically active fragment can be a polypeptide fragment which is,for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360,380, 400, 450, 500, 600 or more contiguous amino acids, including allintegers in between, of a reference polypeptide sequence.

A reference polypeptide may be altered in various ways including aminoacid substitutions, deletions, truncations, and insertions. Methods forsuch manipulations are generally known in the art. For example, aminoacid sequence variants of a reference polypeptide can be prepared bymutations in the DNA. Methods for mutagenesis and nucleotide sequencealterations are well known in the art. See, for example, Kunkel (PNASUSA. 82: 488-492, 1985); Kunkel et al., (Methods in Enzymol. 154:367-382, 1987), U.S. Pat. No. 4,873,192, Watson, J. D. et al.,(“Molecular Biology of the Gene,” Fourth Edition, Benjamin/Cummings,Menlo Park, Calif., 1987) and the references cited therein. Guidance asto appropriate amino acid substitutions that do not affect biologicalactivity of the protein of interest may be found in the model of Dayhoffet al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed.Res. Found., Washington, D.C.).

Methods for screening gene products of combinatorial libraries made bypoint mutations or truncation, and for screening cDNA libraries for geneproducts having a selected property are known in the art. Such methodsare adaptable for rapid screening of the gene libraries generated bycombinatorial mutagenesis of reference polypeptides. Recursive ensemblemutagenesis (REM), a technique which enhances the frequency offunctional mutants in the libraries, can be used in combination with thescreening assays to identify polypeptide variants (Arkin and Yourvan,PNAS USA 89: 7811-7815, 1992; Delgrave et al., Protein Engineering. 6:327-331, 1993). Conservative substitutions, such as exchanging one aminoacid with another having similar properties, may be desirable asdiscussed in more detail below.

Polypeptide variants may contain conservative amino acid substitutionsat various locations along their sequence, as compared to a referenceamino acid sequence. A “conservative amino acid substitution” is one inwhich the amino acid residue is replaced with an amino acid residuehaving a similar side chain. Families of amino acid residues havingsimilar side chains have been defined in the art, which can be generallysub-classified as follows:

Acidic: The residue has a negative charge due to loss of H ion atphysiological pH and the residue is attracted by aqueous solution so asto seek the surface positions in the conformation of a peptide in whichit is contained when the peptide is in aqueous medium at physiologicalpH. Amino acids having an acidic side chain include glutamic acid andaspartic acid.

Basic: The residue has a positive charge due to association with H ionat physiological pH or within one or two pH units thereof (e.g.,histidine) and the residue is attracted by aqueous solution so as toseek the surface positions in the conformation of a peptide in which itis contained when the peptide is in aqueous medium at physiological pH.Amino acids having a basic side chain include arginine, lysine andhistidine.

Charged: The residues are charged at physiological pH and, therefore,include amino acids having acidic or basic side chains (i.e., glutamicacid, aspartic acid, arginine, lysine and histidine).

Hydrophobic: The residues are not charged at physiological pH and theresidue is repelled by aqueous solution so as to seek the innerpositions in the conformation of a peptide in which it is contained whenthe peptide is in aqueous medium. Amino acids having a hydrophobic sidechain include tyrosine, valine, isoleucine, leucine, methionine,phenylalanine and tryptophan.

Neutral/polar: The residues are not charged at physiological pH, but theresidue is not sufficiently repelled by aqueous solutions so that itwould seek inner positions in the conformation of a peptide in which itis contained when the peptide is in aqueous medium. Amino acids having aneutral/polar side chain include asparagine, glutamine, cysteine,histidine, serine and threonine.

This description also characterizes certain amino acids as “small” sincetheir side chains are not sufficiently large, even if polar groups arelacking, to confer hydrophobicity. With the exception of proline,“small” amino acids are those with four carbons or less when at leastone polar group is on the side chain and three carbons or less when not.Amino acids having a small side chain include glycine, serine, alanineand threonine. The gene-encoded secondary amino acid proline is aspecial case due to its known effects on the secondary conformation ofpeptide chains. The structure of proline differs from all the othernaturally-occurring amino acids in that its side chain is bonded to thenitrogen of the α-amino group, as well as the α-carbon. Several aminoacid similarity matrices (e.g., PAM120 matrix and PAM250 matrix asdisclosed for example by Dayhoff et al., (1978), A model of evolutionarychange in proteins. Matrices for determining distance relationships InM. O. Dayhoff, (ed.), Atlas of protein sequence and structure, Vol. 5,pp. 345-358, National Biomedical Research Foundation, Washington D.C.;and by Gonnet et al., (Science. 256: 14430-1445, 1992), however, includeproline in the same group as glycine, serine, alanine and threonine.Accordingly, for the purposes of the present invention, proline isclassified as a “small” amino acid.

The degree of attraction or repulsion required for classification aspolar or nonpolar is arbitrary and, therefore, amino acids specificallycontemplated by the disclosure have been classified as one or the other.Most amino acids not specifically named can be classified on the basisof known behavior.

Amino acid residues can be further sub-classified as cyclic ornon-cyclic, and aromatic or non-aromatic, self-explanatoryclassifications with respect to the side-chain substituent groups of theresidues, and as small or large. The residue is considered small if itcontains a total of four carbon atoms or less, inclusive of the carboxylcarbon, provided an additional polar substituent is present; three orless if not. Small residues are, of course, always non-aromatic.Dependent on their structural properties, amino acid residues may fallin two or more classes. For the naturally-occurring protein amino acids,sub-classification according to this scheme is presented in Table A.

TABLE A Amino acid sub-classification Sub-Classes Amino acids AcidicAspartic acid, Glutamic acid Basic Noncyclic: Arginine, Lysine; Cyclic:Histidine Charged Aspartic acid, Glutamic acid, Arginine, Lysine,Histidine Small Glycine, Serine, Alanine, threonine, ProlinePolar/neutral Asparagine, Histidine, Glutamine, Cysteine, Serine,Threonine Polar/large Asparagine, Glutamine Hydrophobic Tyrosine,Valine, Isoleucine, Leucine, Methionine, Phenylalanine, TryptophanAromatic Tryptophan, Tyrosine, Phenylalanine Residues that influenceGlycine and Proline chain orientation

Conservative amino acid substitution also includes groupings based onside chains. For example, a group of amino acids having aliphatic sidechains is glycine, alanine, valine, leucine, and isoleucine; a group ofamino acids having aliphatic-hydroxyl side chains is serine andthreonine; a group of amino acids having amide-containing side chains isasparagine and glutamine; a group of amino acids having aromatic sidechains is phenylalanine tyrosine, and tryptophan; a group of amino acidshaving basic side chains is lysine, arginine and histidine; and a groupof amino acids having Sulphur-containing side chains is cysteine andmethionine. For example, it is reasonable to expect that replacement ofa leucine with an Isoleucine or valine, an aspartate with a glutamate, athreonine with a serine, or a similar replacement of an amino acid witha structurally related amino acid will not have a major effect on theproperties of the resulting variant polypeptide. Whether an amino acidchange results in a functional truncated and/or variant polypeptide canreadily be determined by assaying its enzymatic activity, as describedherein. Conservative substitutions are shown in Table B under theheading of exemplary substitutions. Amino acid substitutions fallingwithin the scope of the invention, are, in general, accomplished byselecting substitutions that do not differ significantly in their effecton maintaining (a) the structure of the peptide backbone in the area ofthe substitution, (b) the charge or hydrophobicity of the molecule atthe target site, or (c) the bulk of the side chain. After thesubstitutions are Introduced, the variants are screened for biologicalactivity.

TABLE B Exemplary Amino Acid Substitutions Original Preferred ResidueExemplary Substitutions Substitutions Ala Val, Leu, Ile Val Arg Lys,Gln, Asn Lys Asn Gln, His, Lys, Arg Gln Asp Glu Glu Cys Ser Ser Gln Asn,His, Lys, Asn Glu Asp, Lys Asp Gly Pro Pro His Asn, Gln, Lys, Arg ArgIle Leu, Val, Met, Ala, Leu Phe, Norleu Leu Norleu, Ile, Val, Met, IleAla, Phe Lys Arg, Gln, Asn Arg Met Leu, Ile, Phe Leu Phe Leu, Val, Ile,Ala Leu Pro Gly Gly Ser Thr Thr Thr Ser Ser Trp Tyr Tyr Tyr Trp, Phe,Thr, Ser Phe Val Ile, Leu, Met, Phe, Leu Ala, Norleu

Alternatively, similar amino acids for making conservative substitutionscan be grouped into three categories based on the identity of the sidechains. The first group includes glutamic acid, aspartic acid, arginine,lysine, histidine, which all have charged side chains; the second groupincludes glycine, serine, threonine, cysteine, tyrosine, glutamine,asparagine; and the third group includes leucine, isoleucine, valine,alanine, proline, phenylalanine, tryptophan, methionine, as described inZubay, G., Biochemistry, third edition, Wm. C. Brown Publishers (1993).

Thus, a predicted non-essential amino acid residue in referencepolypeptide is typically replaced with another amino acid residue fromthe same side chain family. Alternatively, mutations can be introducedrandomly along all or part of a coding sequence, such as by saturationmutagenesis, and the resultant mutants can be screened for an activityof the parent polypeptide to identify mutants which retain thatactivity. Following mutagenesis of the coding sequences, the encodedpeptide can be expressed recombinantly and the activity of the peptidecan be determined. A “non-essential” amino acid residue is a residuethat can be altered from the wild-type sequence of an embodimentpolypeptide without abolishing or substantially altering one or more ofits activities. Suitably, the alteration does not substantially abolishone of these activities, for example, the activity is at least 20%, 40%,60%, 70% or 80% 100%, 500%, 1000% or more of wild-type. An “essential”amino acid residue is a residue that, when altered from the wild-typesequence of a reference polypeptide, results in abolition of an activityof the parent molecule such that less than 20% of the wild-type activityis present. For example, such essential amino acid residues may includethose that are conserved in reference polypeptides across differentspecies, including those sequences that are conserved in the enzymaticsites of reference polypeptides from various sources.

Accordingly, the present disclosure also contemplates variants of thenaturally-occurring reference polypeptide sequences or theirbiologically-active fragments, wherein the variants are distinguishedfrom the naturally-occurring sequence by the addition, deletion, orsubstitution of one or more amino acid residues. In general, as notedabove, variants will display at least about 30, 40, 50, 55, 60, 65, 70,75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% similarity orsequence identity to a reference polypeptide sequence. Moreover,sequences differing from the native or parent sequences by the addition,deletion, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 or moreamino acids but which retain the properties or activities of a parent orreference polypeptide sequence are contemplated.

In some embodiments, variant polypeptides differ from reference sequenceby at least one but by less than 50, 40, 30, 20, 15, 10, 8, 6, 5, 4, 3or 2 amino acid residue(s). In other embodiments, variant polypeptidesdiffer from a reference sequence by at least 1% but less than 20%, 15%,10% or 5% of the residues. (If this comparison requires alignment, thesequences should be aligned for maximum similarity. “Looped” outsequences from deletions or insertions, or mismatches, are considereddifferences.)

Calculations of sequence similarity or sequence identity betweensequences (the terms are used interchangeably herein) are performed asfollows. To determine the percent identity of two amino acid sequences,or of two nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in one or both of afirst and a second amino acid or nucleic acid sequence for optimalalignment and non-homologous sequences can be disregarded for comparisonpurposes). In certain embodiments, the length of a reference sequencealigned for comparison purposes is at least 30%, preferably at least40%, more preferably at least 50%, 60%, and even more preferably atleast 70%, 80%, 90%, 100% of the length of the reference sequence. Theamino acid residues or nucleotides at corresponding amino acid positionsor nucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position.

The percent identity between the two sequences is a function of thenumber of identical positions shared by the sequences, taking intoaccount the number of gaps, and the length of each gap, which need to beintroduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch, (J.Mol. Biol. 48: 444-453, 1970) algorithm which has been incorporated intothe GAP program in the GCG software package, using either a Blossum 62matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferredembodiment, the percent identity between two nucleotide sequences isdetermined using the GAP program in the GCG software package, using aNWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and alength weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set ofparameters (and the one that should be used unless otherwise specified)are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extendpenalty of 4, and a frameshift gap penalty of 5.

The percent identity between two amino acid or nucleotide sequences canbe determined using the algorithm of E. Meyers and W. Miller (Cabios.4:11-17, 1989) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

The nucleic acid and protein sequences described herein can be used as a“query sequence” to perform a search against public databases to, forexample, identify other family members or related sequences. Suchsearches can be performed using the NBLAST and XBLAST programs (version2.0) of Altschul, et al., (1990, J. Mol. Biol, 215: 403-10). BLASTnucleotide searches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to nucleic acidmolecules of the invention. BLAST protein searches can be performed withthe XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to protein molecules of the invention. To obtaingapped alignments for comparison purposes, Gapped BLAST can be utilizedas described in Altschul et al., (Nucleic Acids Res. 25: 3389-3402,1997). When utilizing BLAST and Gapped BLAST programs, the defaultparameters of the respective programs (e.g., XBLAST and NBLAST) can beused.

In one embodiment, as noted above, polynucleotides and/or polypeptidescan be evaluated using a BLAST alignment tool. A local alignmentconsists simply of a pair of sequence segments, one from each of thesequences being compared. A modification of Smith-Waterman or Sellersalgorithms will find all segment pairs whose scores cannot be improvedby extension or trimming, called high-scoring segment pairs (HSPs). Theresults of the BLAST alignments include statistical measures to indicatethe likelihood that the BLAST score can be expected from chance alone.

The raw score, S, is calculated from the number of gaps andsubstitutions associated with each aligned sequence wherein highersimilarity scores indicate a more significant alignment. Substitutionscores are given by a look-up table (see PAM, BLOSUM).

Gap scores are typically calculated as the sum of G, the gap openingpenalty and L, the gap extension penalty. For a gap of length n, the gapcost would be G+Ln. The choice of gap costs, G and L is empirical, butit is customary to choose a high value for G (10-15), e.g., 11, and alow value for L (1-2) e.g., 1.

The bit score, S′, is derived from the raw alignment score S in whichthe statistical properties of the scoring system used have been takeninto account. Bit scores are normalized with respect to the scoringsystem, therefore they can be used to compare alignment scores fromdifferent searches. The terms “bit score” and “similarity score” areused interchangeably. The bit score gives an indication of how good thealignment is; the higher the score, the better the alignment.

The E-Value, or expected value, describes the likelihood that a sequencewith a similar score will occur in the database by chance. It is aprediction of the number of different alignments with scores equivalentto or better than S that are expected to occur in a database search bychance. The smaller the E-Value, the more significant the alignment. Forexample, an alignment having an E value of e⁻¹¹⁷ means that a sequencewith a similar score is very unlikely to occur simply by chance.Additionally, the expected score for aligning a random pair of aminoacids is required to be negative, otherwise long alignments would tendto have high score independently of whether the segments aligned wererelated. Additionally, the BLAST algorithm uses an appropriatesubstitution matrix, nucleotide or amino acid and for gapped alignmentsuses gap creation and extension penalties. For example, BLAST alignmentand comparison of polypeptide sequences are typically done using theBLOSUM62 matrix, a gap existence penalty of 11 and a gap extensionpenalty of 1.

In one embodiment, sequence similarity scores are reported from BLASTanalyses done using the BLOSUM62 matrix, a gap existence penalty of 11and a gap extension penalty of 1.

In a particular embodiment, sequence identity/similarity scores providedherein refer to the value obtained using GAP Version 10 (GCG, Accelrys,San Diego, Calif.) using the following parameters: % identity and %similarity for a nucleotide sequence using GAP Weight of 50 and LengthWeight of 3, and the nwsgapdna.cmp scoring matrix; % identity and %similarity for an amino acid sequence using GAP Weight of 8 and LengthWeight of 2, and the BLOSUM62 scoring matrix (Henikoff and Henikoff,PNAS USA. 89:10915-10919, 1992). GAP uses the algorithm of Needleman andWunsch (J Mol Biol. 48:443-453, 1970) to find the alignment of twocomplete sequences that maximizes the number of matches and minimizesthe number of gaps.

In one particular embodiment, the variant polypeptide comprises an aminoacid sequence that can be optimally aligned with a reference polypeptidesequence (see, e.g., Sequence Listing) to generate a BLAST bit scores orsequence similarity scores of at least about 50, 60, 70, 80, 90, 100,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230,240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370,380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510,520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650,660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790,800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930,940, 950, 960, 970, 980, 990, 1000, or more, including all integers andranges in between, wherein the BLAST alignment used the BLOSUM62 matrix,a gap existence penalty of 11, and a gap extension penalty of 1.

Variants of a reference polypeptide can be identified by screeningcombinatorial libraries of mutants of a reference polypeptide. Librariesor fragments e.g., N terminal, C terminal, or internal fragments, ofprotein coding sequence can be used to generate a variegated populationof fragments for screening and subsequent selection of variants of areference polypeptide.

Methods for screening gene products of combinatorial libraries made bypoint mutation or truncation, and for screening cDNA libraries for geneproducts having a selected property are known in the art. Such methodsare adaptable for rapid screening of the gene libraries generated bycombinatorial mutagenesis of polypeptides.

The present disclosure also contemplates the use of chimeric or fusionproteins of the reference polypeptides described herein. As used herein,a “chimeric protein” or “fusion protein” includes a referencepolypeptide, or a polypeptide fragment linked to either anotherreference polypeptide (e.g., to create multiple fragments), to anon-reference polypeptide, or to both. In certain embodiments, areference polypeptide can be fused to a heterologous polypeptidesequence. A “heterologous polypeptide” typically has an amino acidsequence corresponding to a protein which is different from thereference protein sequence, and which can be derived from the same or adifferent organism. The reference polypeptide of the fusion protein cancorrespond to all or a portion of a biologically active amino acidsequence.

In certain embodiments, a fusion protein includes at least one or twobiologically active portions of reference protein. The polypeptidesforming the fusion protein are typically linked C-terminus toN-terminus, although they can also be linked C-terminus to C-terminus,N-terminus to N-terminus, or N-terminus to C-terminus. The polypeptidesof the fusion protein can be in any order.

The fusion partner may be designed and included for essentially anydesired purpose provided they do not adversely affect the enzymaticactivity of the polypeptide. For example, in one embodiment, a fusionpartner may comprise a sequence that assists in expressing the protein(an expression enhancer) at higher yields than the native recombinantprotein. Other fusion partners may be selected so as to increase thesolubility or stability of the protein or to enable the protein to betargeted to desired intracellular compartments.

The fusion protein can include a moiety which has a high affinity for aligand. For example, the fusion protein can be a GST-fusion protein inwhich the reference polypeptide sequences are fused to the C-terminus ofthe GST sequences. Such fusion proteins can facilitate the purificationand/or identification of the resulting polypeptide. Alternatively, thefusion protein can be reference polypeptide containing a heterologoussignal sequence at its N-terminus. In certain host cells, expressionand/or secretion of such proteins can be increased through use of aheterologous signal sequence.

Fusion proteins may generally be prepared using standard techniques,described elsewhere herein. A peptide linker sequence may be employed toseparate the first and second polypeptide components by a distancesufficient to ensure that each polypeptide folds into its secondary andtertiary structures, if desired. Exemplary peptide linkers are describedelsewhere herein.

Polynucleotides and Vectors

Embodiments of the present disclosure include polynucleotides encoding adiacylglycerol acyltransferase (DGAT) fusion protein described herein,the fusion protein comprising at least one DGAT polypeptide fused to atleast one intracellular localization domain, such as a bacteriamembrane- or bacterial plasma membrane (PM)-targeting domain. Suchpolynucleotides can be partially or fully isolated from other cellularcomponents, within a vector, for example, a composition comprising sucha vector (e.g., in a tube or kit), or in a host cell, such as modifiedphotosynthetic microorganism.

These polynucleotides and modified photosynthetic microorganismscomprising the same may optionally comprise one or more (Introduced)polynucleotides encoding a lipid biosynthesis protein, and/or one ormore (introduced) polynucleotides encoding a polypeptide associated withglycogen breakdown.

Also included are nucleotide sequences that encode any functionalnaturally-occurring variants or fragments (e.g., allelic variants,orthologs, splice variants) or non-naturally occurring variants orfragments of these native polynucleotides (i.e., optimized byengineering), as well as compositions comprising such polynucleotides,including, for example, cloning and expression vectors.

Also, the modified photosynthetic microorganisms described herein mayoptionally comprise a mutation or deletion in one or more genesassociated with glycogen biosynthesis or storage, alone or incombination with the presence of overexpressed proteins associated withlipid biosynthesis proteins and/or glycogen breakdown. Certain modifiedphotosynthetic microorganisms, for example, for the production of waxesters, may optionally comprise a mutation or deletion in or more genesencoding an aldehyde decarbonylase, an aldehyde dehydrogenase, or both,either alone or in combination with the presence of overexpressedproteins associated with lipid biosynthesis proteins and/or glycogenbreakdown.

The recitations “mutation” or “deletion,” in this context refergenerally to those changes or alterations in a photosyntheticmicroorganism, e.g., a Cyanobacterium, that render the product of thatgene non-functional or having reduced function. Examples of such changesor alterations include nucleotide substitutions, deletions, oradditions/insertions to the coding or regulatory sequences of a targetedgene (e.g., glgA, glgC, pgm, aldehyde decarbonylase, aldehydedehydrogenase), in whole or in part, which disrupt, eliminate,down-regulate, or significantly reduce the expression of the polypeptideencoded by that gene, whether at the level of transcription,translation, post-translational modification, or protein stability. Suchalterations can also reduce the enzymatic activity or other functionalcharacteristic of the protein (e.g., localization), with or withoutreducing expression.

Techniques for producing such alterations or changes, such as byrecombination with a vector having a selectable marker, are exemplifiedherein and known in the molecular biological art. In particularembodiments, one or more alleles of a gene, e.g., two or all alleles,may be mutated or deleted within a photosynthetic microorganism. Inparticular embodiments, modified photosynthetic microorganisms, e.g.,Cyanobacteria, of the present disclosure are merodiploids or partialdiploids.

The “deletion” of a targeted gene or polypeptide may also beaccomplished by targeting the mRNA of that gene, such as by usingvarious antisense technologies (e.g., antisense oligonucleotides andsiRNA) known in the art. Accordingly, targeted genes may be considered“non-functional” when the polypeptide or enzyme encoded by that gene isnot expressed by the modified photosynthetic microorganism, or isexpressed in negligible amounts.

As used herein, the terms “DNA” and “polynucleotide” and “nucleic acid”include a DNA molecule that has been isolated free of total genomic DNAof a particular species. Therefore, a DNA segment encoding a polypeptiderefers to a DNA segment that contains one or more coding sequences yetis substantially isolated away from, or purified free from, totalgenomic DNA of the species from which the DNA segment is obtained.Included within the terms “DNA segment” and “polynucleotide” are DNAsegments and smaller fragments of such segments, and also recombinantvectors, including, for example, plasmids, cosmids, phagemids, phage,viruses, and the like.

As will be understood by those skilled in the art, the polynucleotidesequences of this disclosure can include genomic sequences,extra-genomic and plasmid-encoded sequences and smaller engineered genesegments that express, or may be adapted to express, proteins,polypeptides, peptides and the like. Such segments may be naturallyisolated, or modified synthetically by the hand of man.

Polynucleotides may be single-stranded (coding or antisense) ordouble-stranded, and may be DNA (genomic, cDNA or synthetic) or RNAmolecules. Additional coding or non-coding sequences may, but need not,be present within a polynucleotide of the present invention, and apolynucleotide may, but need not, be linked to other molecules and/orsupport materials.

Polynucleotides may comprise a native sequence (e.g., an endogenoussequence that encodes protein described herein) or may comprise avariant or fragment, or a biological functional equivalent of such asequence. Polynucleotide variants may contain one or more substitutions,additions, deletions and/or insertions, as further described herein,preferably such that the enzymatic activity of the encoded polypeptideis not substantially diminished relative to the unmodified or referencepolypeptide. The effect on the enzymatic activity of the encodedpolypeptide may generally be assessed as described herein and known inthe art.

(i) Intracellular Localization Domain-DGAT Fusion Polynucleotides

Embodiments of the present disclosure Include polynucleotides (e.g.,isolated polynucleotides) that encode any of the intracellularlocalization domain-DGAT fusion proteins described herein, such as themembrane-targeting domain-DGAT fusion proteins. These polynucleotidescomprise at least one sequence encoding a heterologous intracellularlocalization domain described herein, which is fused in-frame to atleast one sequence encoding a DGAT polypeptide, or an active fragment orvariant thereof.

Certain embodiments thus include polynucleotides that encode any one ormore of the intracellular localization domains described herein, wheresuch polynucleotide(s) are fused in-frame to a DGAT-encodingpolynucleotide. Exemplary sequences that encode a membrane-targetingdomain can be found within SEQ ID NOs:200 or 202, the respectivePCC7942-0858 and PCC7942-1015 coding sequences of two methyl-acceptingchemotaxis (MCP) proteins from S. elongatus. For instance, in certainembodiments, the polynucleotide sequence may include about theN-terminal 129 nucleotides of SEQ ID NO:200 (PCC7942-0858), whichencodes the leader sequence of the MCP protein encoded by thePCC7942-0858 gene; additional sequences can also be included, forinstance, about the N-terminal 132, 135, 138, 141, 144, 147, 150, 153,156, 159, 162, 165, 168, 171, 174, 177, 180 or more nucleotides of SEQID NO:200 (PCC7942-0858).

Also included are polynucleotides that encode any one or more of theDGAT polypeptides described herein, where such polynucleotide(s) arefused in-frame to a heterologous intracellular localizationdomain-encoding polynucleotide. In certain embodiments, a DGAT-encodingportion of the fusion protein encodes a DGAT comprising or consisting ofa polypeptide sequence set forth in any one of SEQ ID NOs:58, 59, 60 or61, or a fragment or variant thereof. SEQ ID NO:58 is the sequence ofDGATn; SEQ ID NO: 59 is the sequence of Streptomyces coelicolor DGAT(ScoDGAT or SDGAT); SEQ ID NO:60 is the sequence of Alcanivoraxborkumensis DGAT (AboDGAT); and SEQ ID NO:61 is the sequence of DGATd(Acinetobacter baylii sp.).

In certain embodiments, a DGAT-encoding portion of the fusion proteincomprises or consists of a polynucleotide sequence set forth in any oneof SEQ ID NOs:62, 63, 64, 65 or 66, or a fragment or variant thereof.SEQ ID NO:62 is a codon-optimized for expression in Cyanobacteriasequence that encodes DGATn; SEQ ID NO:63 has homology to SEQ ID NO:62;SEQ ID NO:64 is a codon-optimized for expression in Cyanobacteriasequence that encodes ScoDGAT; SEQ ID NO:65 is a codon-optimized forexpression in Cyanobacteria sequence that encodes AboDGAT; and SEQ IDNO:66 is a codon-optimized for expression in Cyanobacteria sequence thatencodes DGATd. DGATn and DGATd correspond to Acinetobacter baylii DGATand a modified form thereof, which includes two additional amino acidresidues immediately following the initiator methionine.

(ii) Lipid Biosynthesis Genes

In certain embodiments, a modified photosynthetic microorganismcomprises an introduced polynucleotide that encodes one or more lipidbiosynthesis proteins. In some instances, a modified photosyntheticmicroorganism comprises an endogenous polynucleotide that encodes alipid biosynthesis gene, where a regulatory element such as a promoteris introduced upstream of that polynucleotide to regulate or alterexpression of the encoded protein.

In particular embodiments, a modified photosynthetic microorganismcomprises reduced or eliminated expression or activity of a lipidbiosynthesis polypeptide. Included are full or partial deletions, andpoint mutations or insertions of an endogenous lipid biosynthesis genethat reduce or eliminate expression and/or activity of the encodedpolypeptide.

Exemplary lipid biosynthesis genes encode polypeptides such as acylcarrier proteins (ACP), acyl ACP synthases (Aas), acyl-ACP reductases,alcohol dehydrogenases, aldehyde dehydrogenases, aldehydedecarbonylases, thioesterases (TES), acetyl coenzyme A carboxylases(ACCase), phosphatidic acid phosphatases (PAP; or phosphatidatephosphatases), triacylglycerol (TAG) hydrolases, fatty acyl-CoAsynthetases, and lipases/phospholipases, as described herein.

Acyl Carrier Proteins.

In certain embodiments, a modified photosynthetic microorganismcomprises one or more polynucleotides encoding one or more acyl carrierproteins (ACP). Exemplary ACP nucleotide sequences include SEQ ID NO:5from Synechococcus elongatus PCC7942, SEQ ID NOS:7, 9, and 11 fromAcinetobacter sp. ADP1, and SEQ ID NO:13 from Spinacia oleracea.

Acyl ACP Synthases (Aas).

In certain embodiments, a modified photosynthetic microorganismcomprises one or more polynucleotides encoding one or more acyl-ACPsynthetase (Aas) enzymes. In certain embodiments, the Aas nucleotidesequence is derived from the Se918 gene of Synechococcus elongatus. Oneexemplary Aas sequence nucleotide sequence is SEQ ID NO:43 fromSynechococcus elongatus PCC 7942.

In particular embodiments, a modified photosynthetic microorganism ofthe present disclosure has a mutation such as a point mutation,insertion, or full or partial deletion of one or more endogenous Aasgenes, for instance, the Se918 gene of S. elongatus PCC7942, to reduceor eliminate expression and/or activity of the encoded Aas polypeptide.

Acyl-ACP Reductases.

In certain embodiments, a modified photosynthetic microorganismcomprises one or more polynucleotides encoding one or more acyl-ACPreductase polypeptides. Exemplary acyl-ACP reductase nucleotidesequences include orf1594 from Synechococcus elongatus PCC7942 (SEQ IDNO:1), and orfsll0209 from Synechocystis sp. PCC6803 (SEQ ID NO:3).

Alcohol Dehydrogenases.

Certain embodiments may employ one or more alcohol dehydrogenaseencoding polynucleotide sequences. Exemplary alcohol dehydrogenasesinclude slr1192 of Synechocystis sp. PCC6803 (SEQ ID NO:104) andACIAD3612 from Acinetobacter baylii (SEQ ID NO:106).

Aldehyde Dehydrogenases.

Certain embodiments may employ one or more aldehyde dehydrogenaseencoding polynucleotide sequences. Certain embodiments, for example, forthe production of triglycerides or wax esters, may comprise mutationssuch as point mutations, insertions, or full or partial deletions of oneor more endogenous aldehyde dehydrogenase genes. One exemplary aldehydedehydrogenase is orf489 of Synechococcus elongatus PCC7942 (SEQ IDNO:102).

Aldehyde Decarbonylases.

Certain embodiments, for example, for the production of triglycerides orwax esters, may comprise mutations such as point mutations, insertions,or full or partial deletions of one or more endogenous aldehydedecarbonylase genes. One example of an aldehyde decarbonylase is encodedby orf1593 in S. elongatus PCC7942. Another example is an aldehydedecarbonylase encoded by orfsll0208 in Synechocystis sp. PCC6803.

Thloesterases (TES).

In certain embodiments, a modified photosynthetic microorganismcomprises one or more polynucleotides encoding one or more thioesterases(TES) including acyl-ACP thioesterases and/or acyl-CoA thioesterases. Incertain embodiments, the polynucleotide sequence of the TES encodes aTesA or TesB polypeptide from E. coli, or a cytoplasmic TesA variant(*TesA) having the sequence set forth in SEQ ID NO:121.

In certain embodiments, the polynucleotide sequence of the TES comprisesthat of the FatB gene, encoding a FatB enzyme, such as a C8, C12, C14,C16, or C18 FatB enzyme. In certain embodiments, the polynucleotideencodes a thioesterase (e.g., FatB thioesterase), having onlythioesterase activity and little or no lysophospholipase activity. Incertain embodiments, the thioesterase is a FatB acyl-ACP thioesterase,which can hydrolyze acyl-ACP but not acyl-CoA. SEQ ID NO:197 is anexemplary nucleotide sequence of a C8/C10 FatB2 thioesterase derivedfrom Cuphea hookeriana, and SEQ ID NO:122 is codon-optimized forexpression in Cyanobacteria. SEQ ID NO:123 is an exemplary nucleotidesequence of a C12 FatB1 acyl-ACP thioesterase derived from Umbellulariacalifornica, and SEQ ID NO:124 is a codon-optimized version of SEQ IDNO:123 for optimal expression in Cyanobacteria. SEQ ID NO:126 is anexemplary nucleotide sequence of a C14 FatB1 thioesterase derived fromCinnamomum camphora, and SEQ:125 is a codon-optimized version of SEQ IDNO:126. SEQ ID NO:127 is an exemplary nucleotide sequence of a C16 FatB1thioesterase derived from Cuphea hookeriana, and SEQ ID NO:128 is acodon-optimized version of SEQ ID NO:127. In certain embodiments, one ormore FatB sequences are operably linked to a strong promoter, such as aPtrc promoter. In other embodiments, one or more FatB sequences areoperably linked to a relatively weak promoter, such as an arabinosepromoter.

Acetyl Coenzyme A Carboxylases (ACCase).

In certain embodiments, a polynucleotide encodes an acetyl-CoAcarboxylase (ACCase) comprising or consisting of a polypeptide sequenceset forth in any of SEQ ID NOs:55, 45, 46, 47, 48 or 49, or a fragmentor variant thereof. In particular embodiments, a ACCase polynucleotidecomprises or consists of a polynucleotide sequence set forth in any ofSEQ ID NOs:56, 57, 50, 51, 52, 53 or 54, or a fragment or variantthereof. SEQ ID NO:55 is the sequence of Saccharomyces cerevisiaeacetyl-CoA carboxylase (yAcc1); and SEQ ID NO:56 is a codon-optimizedfor expression in Cyanobacteria sequence that encodes yAcc1. SEQ IDNO:45 is Synechococcus sp. PCC 7002 AccA; SEQ ID NO:46 is Synechococcussp. PCC 7002 AccB; SEQ ID NO:47 is Synechococcus sp. PCC 7002 AccC; andSEQ ID NO:48 is Synechococcus sp. PCC 7002 AccD. SEQ ID NO:50 encodesSynechococcus sp. PCC 7002 AccA; SEQ ID NO:51 encodes Synechococcus sp.PCC 7002 AccB; SEQ ID NO:52 encodes Synechococcus sp. PCC 7002 AccC; andSEQ ID NO:53 encodes Synechococcus sp. PCC 7002 AccD. SEQ ID NO:49 is aTriticum aestivum ACCase; and SEQ ID NO:54 encodes this Triticumaestivum ACCase.

Phosphatidic Acid Phosphotases (PAP).

In certain embodiments, a polynucleotide encodes a phosphatidatephosphatase (also referred to as a phosphatidic acid phosphatase; PAP)comprising or consisting of a polypeptide sequence set forth in SEQ IDNO:131, or a fragment or variant thereof. In particular embodiments, aphosphatidate phosphatase polynucleotide comprises or consists of apolynucleotide sequence set forth in SEQ ID NO:129 or SEQ ID NO:130, ora fragment or variant thereof. SEQ ID NO:131 is the sequence ofSaccharomyces cerevisiae phosphatidate phosphatase (yPAH1), and SEQ IDNO:129 is a codon-optimized for expression in Cyanobacteria sequencethat encodes yPAH1. In certain embodiments, the nucleotide sequence ofthe PAP is derived from the E. coli PgpB gene, and/or the PAP gene fromSynechocystis sp. PCC6803.

Triacylglycerol (TAG) Hydrolases.

Certain embodiments employ one or more TAG hydrolase encodingpolynucleotide sequences. Non-limiting examples of TAG hydrolasepolynucleotide sequences include SDP1 (SUGAR-DEPENDENT1) triacylglycerollipase from Arabidopsis thaliana (SEQ ID NO:153), ACIAD1335 fromAcinetobacter sp. ADP1 (SEQ ID NO:154), TG14P from S. cerevisiae (SEQ IDNO:175), and RHA1_ro04722 (YP_704665) TAG lipase from Rhodococcus (SEQID NO:155). Additional polynucleotide sequences for exemplarylipases/esterases include RHA1_ro01602 lipase/esterase from Rhodococcussp. (see SEQ ID NO:156), and the RHA1_ro06856 lipase/esterase (see SEQID NO:119) from Rhodococcus sp.

Fatty Acyl-CoA Synthetases.

Certain embodiments employ one or more fatty acyl-CoA synthetaseencoding polynucleotide sequences. One exemplary fatty acyl-CoAsynthetase includes the FadD gene from E. coli (SEQ ID NO:16) whichencodes a fatty acyl-CoA synthetase having substrate specificity formedium and long chain fatty acids. Other exemplary fatty acyl-CoAsynthetases include those derived from S. cerevisiae; for example, theFaa1p coding sequence is set forth in SEQ ID NO:18, the Faa2p codingsequence is set forth in SEQ ID NO:20, and the Faa3p is set forth in SEQID NO:22. SEQ ID NO:22 is codon-optimized for expression in S. elongatusPCC7942.

Lipases/Phospholipases.

In certain embodiments, a modified photosynthetic microorganismcomprises one or more polynucleotides encoding one or more lipases orphospholipases, including lysophospholipases, or a fragment or variantthereof. In certain embodiments, the encoded lysophospholipase isLysophospholipase L1 (TesA), Lysophospholipase L2, TesB, Vu Patatin 1protein, or a homolog thereof.

In particular embodiments, the encoded phospholipase, e.g., alysophospholipase, is a bacterial phospholipase, or a fragment orvariant thereof, and the polynucleotide comprises a bacterialphospholipase polynucleotide sequence, e.g., a sequence derived fromEscherichia coli, Enterococcus faecalis, or Lactobacillus plantarum. Inparticular embodiments, the encoded phospholipase is LysophospholipaseL1 (TesA), Lysophospholipase L2, TesB, Vu Patatin 1 protein, or afunctional fragment thereof.

In certain embodiments, a lysophospholipase is a bacterialLysophospholipase L1 (TesA) or TesB, such as an E. coliLysophospholipase L1 encoded by a polynucleotide (pldC) having thewild-type sequence set forth in SEQ ID NO:196, or an E. coli TesBencoded by a polynucleotide having the wild-type sequence set forth inSEQ ID NO:132. The polypeptide sequence of E. coli Lysophospholipase L1is provided in SEQ ID NO:133, and the polypeptide sequence of E. coliTesB is provided in SEQ ID NO:134. In other embodiments, alysophospholipase is a Lysophospholipase L2, such as an E. coliLysophospholipase L2 encoded by a polynucleotide (pldB) having thewild-type sequence set forth in SEQ ID NO:135, or a Vu patatin 1 proteinencoded by a polynucleotide having the wild-type sequence set forth inSEQ ID NO:136. The polypeptide sequence of E. coli Lysophospholipase L2is provided in SEQ ID NO:137, and the polypeptide sequence of Vu patatin1 protein is provided in SEQ ID NO:138.

In particular embodiments, the polynucleotide encoding the phospholipasevariant is modified such that it encodes a phospholipase that localizespredominantly to the cytoplasm instead of the periplasm. For example, itmay encode a phospholipase having a deletion or mutation in a regionassociated with periplasmic localization. In particular embodiments, theencoded phospholipase variant is derived from Lysophospholipase L1(TesA). In certain embodiments, the Lysophospholipase L1 (TesA) variantis a bacterial TesA, such as an E. coli Lysophospholipase (TesA) variantencoded by a polynucleotide having the sequence set forth in SEQ IDNO:139. The polypeptide sequence of the Lysophospholipase L1 variant isprovided in SEQ ID NO:121 (PldC(*TesA)).

Additional examples of phospholipase-encoding polynucleotide sequencesinclude phospholipase A1 (PldA) from Acinetobacter sp. ADP1 (SEQ IDNO:140), phospholipase A (PldA) from E. coli (SEQ ID NO:141),phospholipase from Streptomyces coelicolor A3(2) (SEQ ID NO:142),phospholipase A2 (PLA2-α) from Arabidopsis thaliana (SEQ ID NO:143).phospholipase A1/triacylglycerol lipase (DAD1; Defective AntherDehiscence 1) from Arabidopsis thaliana (SEQ ID NO:144), chloroplastDONGLE from Arabidopsis thaliana (SEQ ID NO:145), patatin-like proteinfrom Arabidopsis thaliana (SEQ ID NO:146), and patatin from Anabaenavariabilis ATCC 29413 (SEQ ID NO:147). Additional non-limiting examplesof lysophospholipase-encoding polynucleotide sequences includephospholipase B (Plb1p) from Saccharomyces cerevisiae 5288c (SEQ IDNO:148), phospholipase B (Plb2p) from Saccharomyces cerevisiae S288c(SEQ ID NO:149), ACIAD1057 (TesA homolog) from Acinetobacter ADP1 (SEQID NO:150), ACIAD1943 lysophospholipase from Acinetobacter ADP1 (SEQ IDNO:151), and a lysophospholipase (YP_702320; RHA1_ro02357) fromRhodococcus (SEQ ID NO:152).

(iii) Glycogen Biosynthesis, Storage, and Breakdown Genes

Glycogen Biosynthesis and Storage Genes.

As noted above, certain embodiments include reduced or eliminatedexpression and/or activity of one or more polypeptides associated withglycogen biosynthesis and/or storage, for instance, by mutation of oneor more genes that encode such polypeptides. Included are full orpartial deletions, and point mutations or insertions of one or moreglycogen biosynthesis/storage genes that reduce or eliminate expressionand/or biological activity of the encoded protein(s). Exemplary genesassociated with glycogen synthesis and/or storage include glgC, pgm, andglgA.

Examples of such glgC polynucleotide sequences are provided in SEQ IDNOs:28 (Synechocystis sp. PCC6803), 34 (Nostoc sp. PCC 7120), 33(Anabaena variabilis), 32 (Trichodesmium erythraeum IMS 101), 27(Synechococcus elongatus PCC7942), 30 (Synechococcus sp. WH8102), 31(Synechococcus sp. RCC 307), and 29 (Synechococcus sp. PCC 7002), whichrespectively encode GlgC polypeptides having sequences set forth in SEQID NOs: 86, 87, 88, 89, 90, 91, 92, and 93.

Examples of such pgm polynucleotide sequences are provided in SEQ IDNOs: 25 (Synechocystis sp. PCC6803), 75 (Synechococcus elongatusPCC7942), 26 (Synechococcus sp. WH8102), 78 (Synechococcus RCC307), and80 (Synechococcus 7002), which respectively encode Pgm polypeptideshaving sequences set forth in SEQ ID NOs:74, 76, 77, 79 and 81.

Examples of such glgA polynucleotide sequences are provided in SEQ IDNOs:36 (Synechocystis sp. PCC6803), 42 (Nostoc sp. PCC 7120), 41(Anabaena variabilis), 40 (Trichodesmium erythraeum IMS 101), 35(Synechococcus elongatus PCC7942), 38 (Synechococcus sp. WH8102), 39(Synechococcus sp. RCC 307), and 37 (Synechococcus sp. PCC 7002), whichrespectively encode GlgA polypeptides having sequences set forth in SEQID NOs:94, 95, 96, 97, 98, 99, 100 and 101.

Glycogen Breakdown Genes.

In certain embodiments, a modified photosynthetic microorganism compriseone or more polynucleotides encoding one or more polypeptides associatedwith a glycogen breakdown, or a fragment or variant thereof. Inparticular embodiments, the one or more polypeptides are glycogenphosphorylase (GlgP), glycogen isoamylase (GlgX), glucanotransferase(MalQ), phosphoglucomutase (Pgm), glucokinase (Glk), and/orphosphoglucose isomerase (Pgi), or a functional fragment or variantthereof.

A representative glgP polynucleotide sequence is provided in SEQ IDNO:67, and a representative GlgP polypeptide sequence is provided in SEQID NO:68. A representative glgX polynucleotide sequence is provided inSEQ ID NO:69, and a representative GlgX polypeptide sequence is providedin SEQ ID NO:70. A representative malQ polynucleotide sequence isprovided in SEQ ID NO:71, and a representative MalQ polypeptide sequenceis provided in SEQ ID NO:72. A representative phosphoglucomutase (pgm)polynucleotide sequence is provided in SEQ ID NO:24, and arepresentative phosphoglucomutase (Pgm) polypeptide sequence is providedin SEQ ID NO:73, with others provided infra (SEQ ID NOs:25, 26, 74-81).A representative glk polynucleotide sequence is provided in SEQ IDNO:82, and a representative Glk polypeptide sequence is provided in SEQID NO:83. A representative pgi polynucleotide sequence is provided inSEQ ID NO:84, and a representative Pgi polypeptide sequence is providedin SEQ ID NO:85.

(iv) Polynucleotide Variants, Fragments, Vectors, and Expression Systems

In particular embodiments, a polynucleotide comprises one of thesepolynucleotide sequences, or a fragment or variant thereof, or encodesone of these polypeptide sequences, or a fragment or variant thereof.

Exemplary nucleotide sequences that encode the proteins and enzymes ofthe application encompass full-length reference polynucleotides, as wellas portions of the full-length or substantially full-length nucleotidesequences of these genes or their transcripts or DNA copies of thesetranscripts. Portions of a nucleotide sequence may encode polypeptideportions or segments that retain the biological activity of thereference polypeptide. A portion of a nucleotide sequence that encodes abiologically active fragment of an enzyme provided herein may encode atleast about 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100,120, 150, 200, 300, 400, 500, 600, or more contiguous amino acidresidues, almost up to the total number of amino acids present in afull-length enzyme. It will be readily understood that “intermediatelengths,” in this context and in all other contexts used herein, meansany length between the quoted values, such as 101, 102, 103, etc.; 151,152, 153, etc.; 201, 202, 203, etc.

The polynucleotides described herein, regardless of the length of thecoding sequence itself, may be combined with other DNA sequences, suchas promoters, polyadenylation signals, additional restriction enzymesites, multiple cloning sites, other coding segments, and the like, suchthat their overall length may vary considerably. It is thereforecontemplated that a polynucleotide fragment of almost any length may beemployed, with the total length preferably being limited by the ease ofpreparation and use in the intended recombinant DNA protocol.

The disclosure also contemplates variants of the referencepolynucleotide sequences described herein (see, e.g., the SequenceListing). Nucleic acid variants can be naturally-occurring, such asallelic variants (same locus), homologs (different locus), and orthologs(different organism) or can be non naturally-occurring. Naturallyoccurring variants such as these can be identified and isolated usingwell-known molecular biology techniques including, for example, variouspolymerase chain reaction (PCR) and hybridization-based techniques asknown in the art. Naturally occurring variants can be isolated from anyorganism that encodes one or more genes having an activity of areference polypeptide. Embodiments of the present invention, therefore,encompass Cyanobacteria comprising such naturally-occurringpolynucleotide variants.

Non-naturally occurring variants can be made by mutagenesis techniques,including those applied to polynucleotides, cells, or organisms. Thevariants can contain nucleotide substitutions, deletions, inversions andinsertions. Variation can occur in either or both the coding andnon-coding regions. In certain aspects, non-naturally occurring variantsmay have been optimized for use in Cyanobacteria, such as by engineeringand screening the enzymes for increased activity, stability, or anyother desirable feature.

The variations can produce both conservative and non-conservative aminoacid substitutions (as compared to the originally encoded product). Fornucleotide sequences, conservative variants include those sequencesthat, because of the degeneracy of the genetic code, encode the aminoacid sequence of a reference polypeptide. Variant nucleotide sequencesalso include synthetically derived polynucleotide sequences, such asthose generated, for example, by using site-directed mutagenesis butwhich still encode a biologically active reference polypeptide, asdescribed elsewhere herein. Generally, variants of a particularpolynucleotide sequence will have at least about 30%, 40% 50%, 55%, 60%,65%, 70%, generally at least about 75%, 80%, 85%, 90%, 95% or 98% ormore sequence identity to a reference polynucleotide sequence asdetermined by sequence alignment programs described elsewhere hereinusing default parameters.

Known reference polynucleotide sequences (e.g., described herein) can beused to isolate corresponding sequences and alleles from otherorganisms, particularly other microorganisms. Methods are readilyavailable in the art for the hybridization of nucleic acid sequences.Coding sequences from other organisms may be isolated according to wellknown techniques based on their sequence identity with the codingsequences set forth herein. In these techniques all or part of the knowncoding sequence is used as a probe which selectively hybridizes to otherreference coding sequences present in a population of cloned genomic DNAfragments or cDNA fragments (i.e., genomic or cDNA libraries) from achosen organism.

Accordingly, the present disclosure also contemplates polynucleotidesthat hybridize to reference nucleotide sequences, or to theircomplements, under stringency conditions described below. As usedherein, the term “hybridizes under low stringency, medium stringency,high stringency, or very high stringency conditions” describesconditions for hybridization and washing. Guidance for performinghybridization reactions can be found in Ausubel et al., (1998, supra),Sections 6.3.1-6.3.6. Aqueous and non-aqueous methods are described inthat reference and either can be used.

Reference herein to “low stringency” conditions include and encompassfrom at least about 1% v/v to at least about 15% v/v formamide and fromat least about 1 M to at least about 2 M salt for hybridization at 42°C., and at least about 1 M to at least about 2 M salt for washing at 42°C. Low stringency conditions also may include 1% Bovine Serum Albumin(BSA), 1 mM EDTA, 0.5 M NaHPO₄ (pH 7.2), 7% SDS for hybridization at 65°C., and (i) 2×SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO₄(pH 7.2), 5% SDS for washing at room temperature. One embodiment of lowstringency conditions includes hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 459C, followed by two washes in0.2×SSC, 0.1% SDS at least at 50° C. (the temperature of the washes canbe increased to 55° C. for low stringency conditions).

“Medium stringency” conditions include and encompass from at least about16% v/v to at least about 30% v/v formamide and from at least about 0.5M to at least about 0.9 M salt for hybridization at 42° C., and at leastabout 0.1 M to at least about 0.2 M salt for washing at 55° C. Mediumstringency conditions also may include 1% Bovine Serum Albumin (BSA), 1mM EDTA, 0.5 M NaHPO₄ (pH 7.2), 7% SDS for hybridization at 65° C., and(i) 2×SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO₄ (pH 7.2),5% SDS for washing at 60-65° C. One embodiment of medium stringencyconditions includes hybridizing in 6×SSC at about 459C, followed by oneor more washes in 0.2×SSC, 0.1% SDS at 60° C.

“High stringency” conditions include and encompass from at least about31% v/v to at least about 50% v/v formamide and from about 0.01 M toabout 0.15 M salt for hybridization at 42° C., and about 0.01 M to about0.02 M salt for washing at 55° C. High stringency conditions also mayinclude 1% BSA, 1 mM EDTA, 0.5 M NaHPO₄ (pH 7.2), 7% SDS forhybridization at 65° C., and (i) 0.2×SSC, 0.1% SDS; or (ii) 0.5% BSA, 1mM EDTA, 40 mM NaHPO₄ (pH 7.2), 1% SDS for washing at a temperature inexcess of 65° C. One embodiment of high stringency conditions includeshybridizing in 6×SSC at about 459C, followed by one or more washes in0.2×SSC, 0.1% SDS at 65° C.

In certain embodiments, a reference polypeptide or enzyme describedherein is encoded by a polynucleotide that hybridizes to a disclosednucleotide sequence under very high stringency conditions. Oneembodiment of very high stringency conditions includes hybridizing in0.5 M sodium phosphate, 7% SDS at 65° C., followed by one or more washesin 0.2×SSC, 1% SDS at 65° C.

Other stringency conditions are well known in the art and the skilledartisan will recognize that various factors can be manipulated tooptimize the specificity of the hybridization. Optimization of thestringency of the final washes can serve to ensure a high degree ofhybridization. For detailed examples, see Ausubel et al., supra at pages2.10.1 to 2.10.16 and Sambrook et al. (1989, supra) at sections 1.101 to1.104.

While stringent washes are typically carried out at temperatures fromabout 42° C. to 68° C., one skilled in the art will appreciate thatother temperatures may be suitable for stringent conditions. Maximumhybridization rate typically occurs at about 20° C. to 25° C. below theT_(m) for formation of a DNA-DNA hybrid. It is well known in the artthat the T_(m) is the melting temperature, or temperature at which twocomplementary polynucleotide sequences dissociate. Methods forestimating T_(m) are well known in the art (see Ausubel et al., supra atpage 2.10.8).

In general, the T_(m) of a perfectly matched duplex of DNA may bepredicted as an approximation by the formula: T_(m)=81.5+16.6 (log₁₀M)+0.41 (% G+C)−0.63 (% formamide)−(600/length) wherein: M is theconcentration of Na⁺, preferably in the range of 0.01 molar to 0.4molar; % G+C is the sum of guanosine and cytosine bases as a percentageof the total number of bases, within the range between 30% and 75% G+C;% formamide is the percent formamide concentration by volume; length isthe number of base pairs in the DNA duplex. The T_(m) of a duplex DNAdecreases by approximately 1° C. with every increase of 1% in the numberof randomly mismatched base pairs. Washing is generally carried out atT_(m)−15° C. for high stringency, or T_(m)−30° C. for moderatestringency.

In one example of a hybridization procedure, a membrane (e.g., anitrocellulose membrane or a nylon membrane) containing immobilized DNAis hybridized overnight at 42° C. in a hybridization buffer (50%deionized formamide, 5×SSC, 5× Reinhardt's solution (0.1% fecal, 0.1%polyvinylpyrollidone and 0.1% bovine serum albumin), 0.1% SDS and 200mg/mL denatured salmon sperm DNA) containing a labeled probe. Themembrane is then subjected to two sequential medium stringency washes(i.e., 2×SSC, 0.1% SDS for 15 min at 45° C., followed by 2×SSC, 0.1% SDSfor 15 min at 50° C.), followed by two sequential higher stringencywashes (i.e., 0.2×SSC, 0.1% SDS for 12 min at 55° C. followed by 0.2×SSCand 0.1% SDS solution for 12 min at 65-68° C.).

Polynucleotides and fusions thereof may be prepared, manipulated and/orexpressed using any of a variety of well established techniques knownand available in the art. For example, polynucleotide sequences whichencode polypeptides of the invention, or fusion proteins or functionalequivalents thereof, may be used in recombinant DNA molecules to directexpression of a triglyceride or lipid biosynthesis enzyme in appropriatehost cells. Due to the inherent degeneracy of the genetic code, otherDNA sequences that encode substantially the same or a functionallyequivalent amino acid sequence may be produced and these sequences maybe used to clone and express a given polypeptide.

As will be understood by those of skill in the art, it may beadvantageous in some instances to produce polypeptide-encodingnucleotide sequences possessing non-naturally occurring codons. Forexample, codons preferred by a particular prokaryotic or eukaryotic hostcan be selected to increase the rate of protein expression or to producea recombinant RNA transcript having desirable properties, such as ahalf-life which is longer than that of a transcript generated from thenaturally occurring sequence. Such nucleotides are typically referred toas “codon-optimized.”

Moreover, the polynucleotide sequences described herein can beengineered using methods generally known in the art in order to alterpolypeptide encoding sequences for a variety of reasons, including butnot limited to, alterations which modify the cloning, processing,expression and/or activity of the gene product.

In order to express a desired polypeptide, a nucleotide sequenceencoding the polypeptide, or a functional equivalent, may be insertedinto appropriate expression vector, i.e., a vector that contains thenecessary elements for the transcription and translation of the insertedcoding sequence. Methods which are well known to those skilled in theart may be used to construct expression vectors containing sequencesencoding a polypeptide of interest and appropriate transcriptional andtranslational control elements. These methods include in vitrorecombinant DNA techniques, synthetic techniques, and in vivo geneticrecombination. Such techniques are described in Sambrook et al.,Molecular Cloning, A Laboratory Manual (1989), and Ausubel et al.,Current Protocols in Molecular Biology (1989).

A variety of expression vector/host systems are known and may beutilized to contain and express polynucleotide sequences. In certainembodiments, the polynucleotides of the present disclosure may beintroduced and expressed in Cyanobacterial systems. As such, the presentdisclosure contemplates the use of vector and plasmid systems havingregulatory sequences (e.g., promoters and enhancers) that are suitablefor use in various Cyanobacteria (see, e.g., Koksharova et al., AppliedMicrobiol Biotechnol 58:123-37, 2002). For example, the promiscuousRSF1010 plasmid provides autonomous replication in several Cyanobacteriaof the genera Synechocystis and Synechococcus (see, e.g., Mermet-Bouvieret al., Curr Microbiol 26:323-327, 1993). As another example, the pFC1expression vector is based on the promiscuous plasmid RSF1010. pFC1harbors the lambda c1857 repressor-encoding gene and pR promoter,followed by the lambda cro ribosome-binding site and ATG translationinitiation codon (see, e.g., Mermet-Bouvier et al., Curr Microbiol28:145-148, 1994). The latter is located within the unique NdeIrestriction site (CATATG) of pFC1 and can be exposed after cleavage withthis enzyme for in-frame fusion with the protein-coding sequence to beexpressed.

The “control elements” or “regulatory sequences” present in anexpression vector (or employed separately) are those non-translatedregions of the vector—enhancers, promoters, 5′ and 3′ untranslatedregions—which interact with host cellular proteins to carry outtranscription and translation. Such elements may vary in their strengthand specificity. Depending on the vector system and host utilized, anynumber of suitable transcription and translation elements, includingconstitutive and inducible promoters, may be used. Generally, it iswell-known that strong E. coli promoters work well in Cyanobacteria.Also, when cloning in Cyanobacterial systems, inducible promoters suchas the hybrid lacZ promoter of the PBLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or PSPORT1 plasmid (Gibco BRL, Gaithersburg, Md.) and thelike may be used. Other vectors containing IPTG inducible promoters,such as pAM1579 and pAM2991trc, may be utilized according to the presentinvention.

Certain embodiments may employ a temperature inducible system ortemperature inducible regulatory sequences (e.g., promoters, enhancers,repressors). As one example, an operon with the bacterial phageleft-ward promoter (P_(L)) and a temperature sensitive repressor geneCI857 may be employed to produce a temperature inducible system forproducing fatty acids and/or triglycerides in Cyanobacteria (see, e.g.,U.S. Pat. No. 6,306,639, herein incorporated by reference). It isbelieved that at a non-permissible temperature (low temperature, 30degrees Celsius), the repressor binds to the operator sequence, and thusprevents RNA polymerase from initiating transcription at the PLpromoter. Therefore, the expression of encoded gene or genes isrepressed. When the cell culture is transferred to a permissibletemperature (37-42 degrees Celsius), the repressor cannot bind to theoperator. Under these conditions, RNA polymerase can initiate thetranscription of the encoded gene or genes.

In Cyanobacterial systems, a number of expression vectors or regulatorysequences may be selected depending upon the use intended for theexpressed polypeptide. When large quantities are needed, vectors orregulatory sequences which direct high level expression of encodedproteins may be used. For example, overexpression of ACCase enzymes maybe utilized to increase fatty acid biosynthesis. Such vectors include,but are not limited to, the multifunctional E. coli cloning andexpression vectors such as BLUESCRIPT (Stratagene), in which thesequence encoding the polypeptide of interest may be ligated into thevector in frame with sequences for the amino-terminal Met and thesubsequent 7 residues of β-galactosidase so that a hybrid protein isproduced; pIN vectors (Van Heeke & Schuster, J. Biol. Chem. 264:55035509 (1989)); and the like. pGEX Vectors (Promega, Madison, Wis.) mayalso be used to express foreign polypeptides as fusion proteins withglutathione S-transferase (GST).

Certain embodiments may employ Cyanobacterial promoters or regulatoryoperons. In certain embodiments, a promoter may comprise an rbcLS operonof Synechococcus, as described, for example, in Ronen-Tarazi et al.(Plant Physiology 18:1461-1469, 1995), or a cpc operon of Synechocystissp. strain PCC 6714, as described, for example, in Imashimizu et al. (JBacteriol. 185:6477-80, 2003). In certain embodiments, the tRNApro genefrom Synechococcus may also be utilized as a promoter, as described inChungjatupornchai et al. (Curr Microbial. 38:210-216, 1999). Certainembodiments may employ the nirA promoter from Synechococcus sp. strainPCC7942, which is repressed by ammonium and induced by nitrite (see,e.g., Maeda et al., J. Bacteriol. 180:4080-4088, 1998; and Qi et al.,Applied and Environmental Microbiology 71:5678-5684, 2005). Theefficiency of expression may be increased by the inclusion of enhancerswhich are appropriate for the particular Cyanobacterial cell systemwhich is used, such as those described in the literature.

In certain embodiments, expression vectors or introduced promotersutilized to overexpress an exogenous or endogenous referencepolypeptide, or fragment or variant thereof, comprise a weak promoterunder non-Inducible conditions, e.g., to avoid toxic effects oflong-term overexpression of any of these polypeptides. One example ofsuch a vector for use in Cyanobacteria is the pBAD vector system.Expression levels from any given promoter may be determined, e.g., byperforming quantitative polymerase chain reaction (qPCR) to determinethe amount of transcript or mRNA produced by a promoter, e.g., beforeand after induction. In certain instances, a weak promoter is defined asa promoter that has a basal level of expression of a gene or transcriptof interest, in the absence of inducer, that is ≤2.0% of the expressionlevel produced by the promoter of the rnpB gene in S. elongatus PCC7942.In other embodiments, a weak promoter is defined as a promoter that hasa basal level of expression of a gene or transcript of interest, in theabsence of inducer, that is ≤5.0% of the expression level produced bythe promoter of the rnpB gene in S. elongatus PCC7942.

It will be apparent that further to their use in vectors, any of theregulatory elements described herein (e.g., promoters, enhancers,repressors, ribosome binding sites, transcription termination sites) maybe introduced directly into the genome of a photosynthetic microorganism(e.g., Cyanobacterium), typically in a region surrounding (e.g.,upstream or downstream of) an endogenous or naturally-occurringreference gene/polynucleotide sequence described herein, to regulateexpression (e.g., facilitate overexpression) of that gene.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding a polypeptide of interest. Suchsignals include the ATG initiation codon and adjacent sequences. Incases where sequences encoding the polypeptide, its initiation codon,and upstream sequences are inserted into the appropriate expressionvector, no additional transcriptional or translational control signalsmay be needed. However, in cases where only coding sequence, or aportion thereof, is inserted, exogenous translational control signalsincluding the ATG initiation codon should be provided. Furthermore, theinitiation codon should be in the correct reading frame to ensuretranslation of the entire insert. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.

A variety of protocols for detecting and measuring the expression ofpolynucleotide-encoded products, using either polyclonal or monoclonalantibodies specific for the product are known in the art. Examplesinclude enzyme-linked immunosorbent assay (ELISA), radioimmunoassay(RIA), and fluorescence activated cell sorting (FACS). These and otherassays are described, among other places, in Hampton et al., SerologicalMethods, a Laboratory Manual (1990) and Maddox et al., J. Exp. Med.158:1211-1216 (1983). The presence or expression levels of a desiredpolynucleotide may also be confirmed by PCR.

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides include oligolabeling,nick translation, end-labeling or PCR amplification using a labelednucleotide. Alternatively, the sequences, or any portions thereof may becloned into a vector for the production of an mRNA probe. Such vectorsare known in the art, are commercially available, and may be used tosynthesize RNA probes in vitro by addition of an appropriate RNApolymerase such as T7, T3, or SP6 and labeled nucleotides. Theseprocedures may be conducted using a variety of commercially availablekits. Suitable reporter molecules or labels, which may be used includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, cofactors, inhibitors, magnetic particles,and the like.

Cyanobacterial host cells transformed with a polynucleotide sequence ofinterest may be cultured under conditions suitable for the expressionand recovery of the protein from cell culture. The protein produced by arecombinant cell may be secreted or contained intracellularly dependingon the sequence and/or the vector used. As will be understood by thoseof skill in the art, expression vectors containing polynucleotides ofthe disclosure may be designed to contain signal sequences which directlocalization of the encoded polypeptide to a desired site within thecell. Other recombinant constructions may be used to join sequencesencoding a polypeptide of interest to nucleotide sequence encoding apolypeptide domain which will direct secretion of the encoded protein.

Modified Photosynthetic Microorganisms

Certain embodiments relate to modified photosynthetic microorganisms,including Cyanobacteria, and methods of use thereof, wherein themodified photosynthetic microorganisms comprise one or moreover-expressed, exogenous or introduced intracellular localizationdomain-DGAT fusion proteins, and a corresponding polynucleotide thatencodes the same, where the DGAT fusion protein comprises a heterologousintracellular localization domain and a DGAT polypeptide, or variant orfragment thereof. In particular embodiments, the DGAT fusion protein isa membrane-targeting domain- or plasma membrane (PM)-targetingdomain-DGAT fusion proteins. In some embodiments, the DGAT polypeptidevariant or fragment retains at least 50% of one or more activities ofthe wild-type DGAT polypeptide.

In certain aspects, the DGAT-fusion protein-expressing photosyntheticmicroorganisms described herein can further comprise one or moreintroduced or overexpressed lipid biosynthesis proteins. Examples oflipid biosynthesis proteins include, without limitation, acyl carrierproteins (ACP), acyl ACP synthases (Aas), acyl-ACP reductases, alcoholdehydrogenases, aldehyde dehydrogenases, aldehyde decarbonylases,thioesterases (TES), acetyl coenzyme A carboxylases (ACCase),phosphatidic acid phosphatases (PAP; or phosphatidate phosphatases),triacylglycerol (TAG) hydrolases, fatty acyl-CoA synthetases, andlipases/phospholipases, including any combinations thereof.

Certain preferred combinations include modified photosyntheticmicroorganisms having an exogenous or overexpressed DGAT fusion proteindescribed herein in combination with an exogenous or overexpressed ACP;a DGAT fusion protein in combination with an Aas; a DGAT fusion proteinin combination with an ACP and an Aas; a DGAT fusion protein incombination with an ACP and a TES such as *TesA or a FatB; a DGAT fusionprotein in combination with an Aas and a TES such as *TesA or a FatB;and/or a DGAT fusion protein in combination with an ACP, an Aas, and aTES.

Also included are combinations that incorporate one or more TAGhydrolases into a TAG-producing strain. For example, certain embodimentsinclude modified photosynthetic microorganisms having a DGAT fusionprotein described herein, an exogenous or overexpressed ACP, Aas, orboth, in combination with an exogenous or over-expressed TAG hydrolase,and optionally a TES. Certain embodiments, however, may employ a DGATfusion protein and an over-expressed or exogenous TAG hydrolase, andoptionally a TES, such as TesA (or *TesA) or any one or more of the FatBsequences, with or without an ACP or Aas. Hence, these and relatedembodiments may be employed separately from those that require an ACP,an Aas, or both. For instance, certain embodiments may comprise a DGATfusion protein and TAG hydrolase, and optionally a TES. Any one of theseembodiments can be further combined with one or more additional lipidbiosynthesis proteins, such as an ACCase, a PAP, a fatty acyl-CoAsynthetase, and/or a PL such as PLC.

Certain combinations incorporate one or more fatty acyl-CoA synthetases(e.g., FadD) into a DGAT fusion protein-expressing photosyntheticmicroorganism. For instance, certain embodiments include modifiedphotosynthetic microorganisms having an exogenous or overexpressed ACP,Aas, or both, in combination with a DGAT fusion protein and a fattyacyl-CoA synthetase, and optionally a TES and/or a TAG hydrolase.Certain embodiments, however, may employ a DGAT fusion protein and anover-expressed or exogenous fatty acyl-CoA synthetase, and optionally aTES, such as TesA (or *TesA) or any one or more of the FatB sequences,with or without an ACP or Aas. Hence, these and related embodiments maybe employed separately from those that require an ACP, Aas, or both. Forinstance, certain embodiments may comprise a DGAT fusion protein and afatty acyl-CoA synthetase, and optionally a TES (e.g., TesA, FatB). Anyone of these embodiments can be further combined with one or moreadditional lipid biosynthesis proteins, such as an ACCase, a PAP, a TAGhydrolase, and/or a PL such as PLC.

Any one of these embodiments can also be combined with one or moreintroduced or overexpressed polynucleotides encoding a protein involvedin a glycogen breakdown pathway, and/or with a strain having reducedexpression of glycogen biosynthesis or storage pathways (e.g., full orpartial deletion of glucose-1-phosphate adenylyltransferase (glgC) geneand/or a phosphoglucomutase (pgm) gene). For instance, a specificmodified photosynthetic microorganism could comprise a DGAT fusionprotein described herein, an exogenous or overexpressed ACP, Aas, DGATand PAP, combined with a full or partial deletion of the glgC geneand/or the pgm gene.

Photosynthetic microorganisms of the present disclosure can also bemodified to increase the production of fatty acids by introducing one ormore exogenous polynucleotide sequences that encode one or more enzymesassociated with fatty acid synthesis. In certain aspects, the exogenouspolynucleotide sequence encodes an enzyme that comprises an acetyl-CoAcarboxylase (ACCase) activity, typically allowing increased ACCaseexpression, and, thus, increased intracellular ACCase activity.Increased intracellular ACCase activity contributes to the increasedproduction of fatty acids because this enzyme catalyzes the “commitmentstep” of fatty acid synthesis. Similarly, in some aspects, modifiedphotosynthetic microorganisms may comprise a DGAT fusion proteindescribed herein in combination with an acyl-ACP reductase, forinstance, to increase the production of fatty acids, a starting materialfor triglycerides, and thereby increase production of triglycerides.

Other combinations include, for example, a modified photosyntheticmicroorganism comprising a DGAT fusion protein described herein and oneof the following: an exogenous or overexpressed ACP in combination withan exogenous or overexpressed ACCase; an Aas in combination with anACCase; an ACP and an Aas in combination with an ACCase; an ACP incombination with a PAP; an Aas in combination with a PAP; an ACP and anAas in combination with a PAP; an ACP in combination with a PL such asPLA, PLB, or PLC; an Aas in combination with a PL; and an ACP and an Aasin combination with a PL. Any one of these embodiments can be combinedwith each other (e.g., ACP, Aas, ACCase, and PAP), and/or furthercombined with an exogenous or overexpressed TES. Any one of theseembodiments can also be combined with one or more introducedpolynucleotides encoding a protein involved in a glycogen breakdownpathway, and/or with a strain having reduced expression of glycogenbiosynthesis or storage pathways (e.g., full or partial deletion ofglucose-1-phosphate adenylyltransferase (glgC) gene and/or aphosphoglucomutase (pgm) gene).

Any one of the above embodiments can also be combined with a strainhaving reduced expression of an aldehyde decarbonylase. In certainembodiments, such as Cyanobacteria including S. elongatus PCC7942,orf1593 resides directly upstream of orf1594 (acyl-ACP reductase codingregion) and encodes an aldehyde decarbonylase. According to onenon-limiting theory, because the aldehyde decarbonylase encoded byorf1593 utilizes acyl aldehyde as a substrate for alkane production,reducing expression of this protein may further increase yields of freefatty acids by shunting acyl aldehydes (produced by acyl-ACP reductase)away from an alkane-producing pathway, and towards a fattyacid-producing and storage pathway. PCC7942_orf1593 orthologs can befound, for example, in Synechocystis sp. PCC6803 (encoded byorfsll0208), N. punctiforme PCC 73102, Thermosynechococcus elongatusBP-1, Synechococcus sp. Ja-3-3AB, P. marinus MIT9313, P. marinus NATL2A,and Synechococcus sp. RS 9117, the latter having at least two paralogs(RS 9117-1 and -2). Included are strains having mutations or full orpartial deletions of one or more genes encoding these and other aldehydedecarbonylases, such as S. elongatus PCC7942 having a full or partialdeletion of orf1593, and Synechocystis sp. PCC6803 having a full orpartial deletion of orfsll0208. For instance, a specific modifiedphotosynthetic microorganism could comprise an overexpressed acyl-ACPreductase, combined with a full or partial deletion of the glgC geneand/or the pgm gene, optionally combined with an overexpressed ACP,ACCase, DGAT/acyl-CoA synthetase, or all of the foregoing, andoptionally combined with a full or partial deletion of a gene encodingan aldehyde decarbonylase (e.g., PCC7942_orf1593, PCC6803_orfsll0208).

Any one of these embodiments can also be combined with a strain havingreduced expression of an acyl-ACP synthetase (Aas). Without wishing tobe bound by any one theory, an endogenous aldehyde dehydrogenase isacting on the acyl-aldehydes generated by orf1594 and converting them tofree fatty acids. The normal role of such a dehydrogenase might involveremoving or otherwise dealing with damaged lipids. In this scenario, itis then likely that the Aas gene product recycles these free fatty acidsby ligating them to ACP. Accordingly, reducing or eliminating expressionof the Aas gene product might ultimately increase production of fattyacids, by reducing or preventing their transfer to ACP. Included aremutations and full or partial deletions of one or more Aas genes, suchas the Aas gene of Synechococcus elongatus PCC 7942. As one example, aspecific modified photosynthetic microorganism could comprise anoverexpressed acyl-ACP reductase, combined with a full or partialdeletion of the glgC gene and/or the pgm gene, optionally combined withan overexpressed ACP, ACCase, DGAT/acyl-CoA synthetase, or all of theforegoing, optionally combined with a full or partial deletion of a geneencoding an aldehyde decarbonylase (e.g., PCC7942_orf1593,PCC6803_orfsll0208), and optionally combined with a full or partialdeletion of an Aas gene encoding an acyl-ACP synthetase.

Any one or more of these embodiments can also be combined with a strainhaving increased expression of an aldehyde dehydrogenase. One exemplaryaldehyde dehydrogenase is encoded by orf0489 of Synechococcus elongatusPCC7942. Also included are homologs or paralogs thereof, functionalequivalents thereof, and fragments or variants thereof. Functionalequivalents can include aldehyde dehydrogenases with the ability toconvert acyl aldehydes (e.g., nonyl-aldehyde) into fatty acids. Inspecific embodiments, the aldehyde dehydrogenase has the amino acidsequence of SEQ ID NO:103 (encoded by the polynucleotide sequence of SEQID NO:102), or an active fragment or variant of this sequence.

Some modified photosynthetic microorganisms may comprise a DGAT fusionprotein described herein and an introduced or overexpressed acyl-ACPreductase, to increase production of triglycerides; optionally infurther combination with an introduced or overexpressed alcoholdehydrogenase, for instance, to produce wax esters relative to otherlipids. Certain of these and related embodiments may be combined withreduced expression and/or activity of at least one endogenous aldehydedecarbonylase, endogenous aldehyde dehydrogenase, or both.

For instance, particular modified photosynthetic microorganisms maycomprise a DGAT fusion protein described herein in combination with anoverexpressed or introduced acyl-ACP reductase and an overexpressed orintroduced alcohol dehydrogenase, and in further combination with atleast one mutation (e.g., point mutation, insertion, full or partialdeletion) that reduces the expression and/or activity of an endogenousaldehyde decarbonylase. Certain modified photosynthetic microorganismsmay comprise a DGAT fusion protein in combination with an overexpressedor introduced acyl-ACP reductase and an overexpressed or introducedalcohol dehydrogenase, and in further combination with at least onemutation (e.g., point mutation, insertion, full or partial deletion)that reduces the expression and/or activity of an endogenous aldehydedehydrogenase. Some embodiments may include modified photosyntheticmicroorganisms that comprises a DGAT fusion protein in combination withan overexpressed or introduced acyl-ACP reductase and an overexpressedor introduced alcohol dehydrogenase, in further combination with atleast one mutation that reduces the expression and/or activity of anendogenous aldehyde dehydrogenase and at least one mutation that reducesthe expression and/or activity of an endogenous aldehyde decarbonylase.In specific embodiments, for instance, where the modified photosyntheticmicroorganism is S. elongatus, the aldehyde dehydrogenase is encoded byorf0489 and the aldehyde decarbonylase is encoded by orf1593 of S.elongatus.

Other combinations include, for example, a modified photosyntheticmicroorganism comprising a DGAT fusion protein described herein andreduced glycogen accumulation, in combination with one more of anoverexpressed ACP; an overexpressed acyl-ACP reductase in combinationwith an overexpressed ACP; an overexpressed acyl-ACP reductase incombination with an overexpressed ACCase; an overexpressed acyl-ACPreductase in combination with an overexpressed ACP and an overexpressedACCase; an overexpressed acyl-ACP reductase in combination with anoverexpressed acyl-CoA synthetase (e.g., a membrane-targetingdomain-DGAT fusion/acyl-CoA synthetase combination); an overexpressedacyl-ACP reductase with an overexpressed ACCase optionally incombination with an overexpressed acyl-CoA synthetase; and anoverexpressed acyl-ACP reductase with an overexpressed ACP and ACCase,optionally in combination with an overexpressed acyl-CoA synthetase.Acyl-ACP reductase and DGAT-overexpressing strains, optionally incombination with an overexpressed acyl-CoA synthetase, typically produceincreased triglycerides relative to DGAT-only overexpressing strains.Any one of these embodiments can be combined with one or more introducedpolynucleotides encoding a protein involved in a glycogen breakdownpathway, and/or with a strain having reduced expression of glycogenbiosynthesis or storage pathways (e.g., full or partial deletion ofglucose-1-phosphate adenylyltransferase (glgC) gene and/or aphosphoglucomutase (pgm) gene). The present disclosure contemplates theuse of any type of polynucleotide encoding a protein or enzymeassociated with glycogen breakdown, removal, and/or elimination, as longas the modified photosynthetic microorganism accumulates a reducedamount of glycogen as compared to the wild type photosyntheticmicroorganism.

Increased expression or overexpression can be achieved a variety ofways, for example, by introducing a polynucleotide into themicroorganism, modifying an endogenous gene to overexpress thepolypeptide (e.g., by introducing an exogenous regulatory element suchas a promoter), or both. For instance, one or more copies of anotherwise endogenous polynucleotide sequence can be introduced byrecombinant techniques to increase expression, that is, to createadditional copies of the otherwise endogenous polynucleotide sequence.Decreased expression and/or activity can also be achieved a variety ofways, described elsewhere herein and known in the art, including bymutation of coding and/or regulatory sequences of a gene of interest,and/or by RNA inhibition.

Modified photosynthetic microorganisms of the present disclosure may beproduced using any type of photosynthetic microorganism. These include,but are not limited to photosynthetic bacteria, green algae, andcyanobacteria. The photosynthetic microorganism can be, for example, anaturally photosynthetic microorganism, such as a Cyanobacterium, or anengineered photosynthetic microorganism, such as an artificiallyphotosynthetic bacterium. Exemplary microorganisms that are eithernaturally photosynthetic or can be engineered to be photosyntheticinclude, but are not limited to, bacteria; fungi; archaea; protists;eukaryotes, such as a green algae; and animals such as plankton,planarian, and amoeba. Examples of naturally occurring photosyntheticmicroorganisms include, but are not limited to, Spirulina maximum,Spirulina platensis, Dunaliella salina, Botrycoccus braunii, Chlorellavulgaris, Chlorella pyrenoidosa, Serenastrum capricomutum, Scenedesmusauadricauda, Porphyridium cruentum, Scenedesmus acutus, Dunaliella sp.,Scenedesmus obliquus, Anabaenopsis, Aulosira, Cylindrospermum,Synechococcus sp., Synechocystis sp., and/or Tolypothrix.

A modified Cyanobacteria of the present disclosure may be from anygenera or species of Cyanobacteria that is genetically manipulable,i.e., permissible to the introduction and expression of exogenousgenetic material. Examples of Cyanobacteria that can be engineeredaccording to the methods of the present disclosure include, but are notlimited to, the genus Synechocystis, Synechococcus, Thermosynechococcus,Nostoc, Prochlorococcus, Microcystis, Anabaena, Spirulina, andGloeobacter.

Cyanobacteria, also known as blue-green algae, blue-green bacteria, orCyanophyta, is a phylum of bacteria that obtain their energy throughphotosynthesis. Cyanobacteria can produce metabolites, such ascarbohydrates, proteins, lipids and nucleic acids, from CO₂, water,inorganic salts and light. Any Cyanobacteria may be used according tothe present invention.

Cyanobacteria include both unicellular and colonial species. Coloniesmay form filaments, sheets or even hollow balls. Some filamentouscolonies show the ability to differentiate into several different celltypes, such as vegetative cells, the normal, photosynthetic cells thatare formed under favorable growing conditions; akinetes, theclimate-resistant spores that may form when environmental conditionsbecome harsh; and thick-walled heterocysts, which contain the enzymenitrogenase, vital for nitrogen fixation.

Heterocysts may also form under the appropriate environmental conditions(e.g., anoxic) whenever nitrogen is necessary. Heterocyst-formingspecies are specialized for nitrogen fixation and are able to fixnitrogen gas, which cannot be used by plants, into ammonia (NH₃),nitrites (NO₂), or nitrates (NO₃), which can be absorbed by plants andconverted to protein and nucleic acids.

Many Cyanobacteria also form motile filaments, called hormogonia, whichtravel away from the main biomass to bud and form new colonieselsewhere. The cells in a hormogonium are often thinner than in thevegetative state, and the cells on either end of the motile chain may betapered. In order to break away from the parent colony, a hormogoniumoften must tear apart a weaker cell in a filament, called a necridium.

Each individual Cyanobacterial cell typically has a thick, gelatinouscell wall. Cyanobacteria differ from other gram-negative bacteria inthat the quorum sensing molecules autoinducer-2 and acyl-homoserinelactones are absent. They lack flagella, but hormogonia and someunicellular species may move about by gliding along surfaces. In watercolumns, some Cyanobacteria float by forming gas vesicles, like inarchaea.

Cyanobacteria have an elaborate and highly organized system of internalmembranes that function in photosynthesis. Photosynthesis inCyanobacteria generally uses water as an electron donor and producesoxygen as a by-product, though some Cyanobacteria may also use hydrogensulfide, similar to other photosynthetic bacteria. Carbon dioxide isreduced to form carbohydrates via the Calvin cycle. In most forms, thephotosynthetic machinery is embedded into folds of the cell membrane,called thylakoids. Due to their ability to fix nitrogen in aerobicconditions, Cyanobacteria are often found as symbionts with a number ofother groups of organisms such as fungi (e.g., lichens), corals,pteridophytes (e.g., Azolla), and angiosperms (e.g., Gunnera), amongothers.

Cyanobacteria are the only group of organisms that are able to reducenitrogen and carbon in aerobic conditions. The water-oxidizingphotosynthesis is accomplished by coupling the activity of photosystem(PS) II and I (Z-scheme). In anaerobic conditions, Cyanobacteria arealso able to use only PS I (i.e., cyclic photophosphorylation) withelectron donors other than water (e.g., hydrogen sulfide, thiosulphate,or molecular hydrogen), similar to purple photosynthetic bacteria.Furthermore, Cyanobacteria share an archaeal property; the ability toreduce elemental sulfur by anaerobic respiration in the dark. TheCyanobacterial photosynthetic electron transport system shares the samecompartment as the components of respiratory electron transport.Typically, the plasma membrane contains only components of therespiratory chain, while the thylakoid membrane hosts both respiratoryand photosynthetic electron transport.

Phycobilisomes, attached to the thylakoid membrane, act as lightharvesting proteins for the photosystems of Cyanobacteria. Thephycobilisome components (phycobiliproteins) are responsible for theblue-green pigmentation of most Cyanobacteria. Color variations aremainly due to carotenoids and phycoerythrins, which may provide thecells with a red-brownish coloration. In some Cyanobacteria, the colorof light influences the composition of phycobilisomes. In green light,the cells accumulate more phycoerythrin, whereas in red light theyproduce more phycocyanin. Thus, the bacteria appear green in red lightand red in green light. This process is known as complementary chromaticadaptation and represents a way for the cells to maximize the use ofavailable light for photosynthesis.

In particular embodiments, the Cyanobacteria may be, e.g., a marine formof Cyanobacteria or a freshwater form of Cyanobacteria. Examples ofmarine forms of Cyanobacteria include, but are not limited toSynechococcus WH8102, Synechococcus RCC307, Synechococcus NKBG 15041c,and Trichodesmium. Examples of freshwater forms of Cyanobacteriainclude, but are not limited to, S. elongatus PCC 7942, SynechocystisPCC 6803, Plectonema boryanum, and Anabaena sp. Exogenous geneticmaterial encoding the desired enzymes or polypeptides may be Introducedeither transiently, such as in certain self-replicating vectors, orstably, such as by integration (e.g., recombination) into theCyanobacterium's native genome.

In other embodiments, a genetically modified Cyanobacteria of thepresent disclosure may be capable of growing in brackish or salt water.When using a freshwater form of Cyanobacteria, the overall net cost forproduction of triglycerides will depend on both the nutrients requiredto grow the culture and the price for freshwater. One can foreseefreshwater being a limited resource in the future, and in that case itwould be more cost effective to find an alternative to freshwater. Twosuch alternatives include: (1) the use of waste water from treatmentplants; and (2) the use of salt or brackish water.

Salt water in the oceans can range in salinity between 3.1% and 3.8%,the average being 3.5%, and this is mostly, but not entirely, made up ofsodium chloride (NaCl) ions. Brackish water, on the other hand, has moresalinity than freshwater, but not as much as seawater. Brackish watercontains between about 0.5% and 3% salinity, and thus includes a largerange of salinity regimes and is therefore not precisely defined. Wastewater is any water that has undergone human influence. It consists ofliquid waste released from domestic and commercial properties, industry,and/or agriculture and can encompass a wide range of possiblecontaminants at varying concentrations.

There is a broad distribution of Cyanobacteria in the oceans, withSynechococcus filling just one niche. Specifically, Synechococcus sp.PCC 7002 (formerly known as Agmenellum quadruplicatum strain PR-6) growsin brackish water, is unicellular and has an optimal growing temperatureof 38° C. While this strain is well suited to grow in conditions of highsalt, it will grow slowly in freshwater. In particular embodiments, thepresent disclosure contemplates the use of a Cyanobacteria S. elongatusPCC 7942, altered in a way that allows for growth in either waste wateror salt/brackish water. A S. elongatus PCC 7942 mutant resistant tosodium chloride stress has been described (Bagchi, S. N. et al.,Photosynth Res. 2007, 92:87-101), and a genetically modified S.elongatus PCC 7942 tolerant of growth in salt water has been described(Waditee, R. et al., PNAS. 2002, 99:4109-4114). According to the presentinvention, a salt water tolerant strain is capable of growing in wateror media having a salinity in the range of 0.5% to 4.0% salinity,although it is not necessarily capable of growing in all salinitiesencompassed by this range. In one embodiment, a salt tolerant strain iscapable of growth in water or media having a salinity in the range of1.0% to 2.0% salinity. In another embodiment, a salt water tolerantstrain is capable of growth in water or media having a salinity in therange of 2.0% to 3.0% salinity.

Examples of Cyanobacteria that may be utilized and/or geneticallymodified according to the methods described herein include, but are notlimited to, Chroococcales Cyanobacteria from the genera Aphanocapsa,Aphanothece, Chamaesiphon, Chroococcus, Chroogloeocystis,Coelosphaerium, Crocosphaera, Cyanobacterium, Cyanobium, Cyanodictyon,Cyanosarcina, Cyanothece, Dactylococcopsis, Gloecapsa, Gloeothece,Merismopedia, Microcystis, Radiocystis, Rhabdoderma, Snowella,Synychococcus, Synechocystis, Thermosenechococcus, and Woronichinia;Nostacales Cyanobacteria from the genera Anabaena, Anabaenopsis,Aphanizomenon, Aulosira, Colothrix, Coleodesmium, Cyanospira,Cylindrospermosis, Cylindrospermum, Fremyella, Gleotrichia, Microchaete,Nodularia, Nostoc, Rexia, Richelia, Scytonema, Sprirestis, andToypothrix; Oscillatoriales Cyanobacteria from the genera Arthrospira,Geitlerinema, Halomicronema, Halospirulina, Katagnymene, Leptolyngbya,Limnothrix, Lyngbya, Microcoleus, Oscillatoria, Phormidium,Planktothricoides, Planktothrix, Plectonema, Pseudoanabaena/Limnothrix,Schizothrix, Spirulina, Symploca, Trichodesmium, Tychonema;Pleurocapsales cyanobacterium from the genera Chroococcidiopsis,Dermocarpa, Dermocarpella, Myxosarcina, Pleurocapsa, Stanieria,Xenococcus; Prochlorophytes Cyanobacterium from the genera Prochloron,Prochlorococcus, Prochlorothrix; and Stigonematales cyanobacterium fromthe genera Capsosira, Chlorogeoepsis, Fischerella, Hapalosiphon,Mastigocladopsis, Nostochopsis, Stigonema, Symphyonema, Symphonemopsis,Umezakia, and Westiellopsis. In certain embodiments, the Cyanobacteriumis from the genus Synechococcus, including, but not limited toSynechococcus bigranulatus, Synechococcus elongatus, Synechococcusleopoliensis, Synechococcus lividus, Synechococcus nidulans, andSynechococcus rubescens.

In certain embodiments, the Cyanobacterium is Anabaena sp. strain PCC7120, Synechocystis sp. strain PCC 6803, Nostoc muscorum, Nostocellipsosporum, or Nostoc sp. strain PCC 7120. In certain preferredembodiments, the Cyanobacterium is S. elongatus sp. strain PCC 7942.

Additional examples of Cyanobacteria that may be utilized in the methodsprovided herein include, but are not limited to, Synechococcus sp.strains WH7803, WH8102, WH8103 (typically genetically modified byconjugation), Baeocyte-forming Chroococcidiopsis spp. (typicallymodified by conjugation/electroporation), non-heterocyst-formingfilamentous strains Planktothrix sp., Plectonema boryanum M101(typically modified by electroporation), and Heterocyst-forming strainsAnabaena sp. strains ATCC 29413 (typically modified by conjugation),Tolypothrix sp. strain PCC 7601 (typically modified byconjugation/electroporation) and Nostoc punctiforme strain ATCC 29133(typically modified by conjugation/electroporation).

In certain preferred embodiments, the Cyanobacterium may be S. elongatussp. strain PCC 7942 or Synechococcus sp. PCC 7002 (originally known asAgmenellum quadruplicatum).

In particular embodiments, the genetically modified, photosyntheticmicroorganism, e.g., Cyanobacteria, of the present disclosure may beused to produce triglycerides and/or other carbon-based products fromjust sunlight, water, air, and minimal nutrients, using routine culturetechniques of any reasonably desired scale. In certain embodiments, thepresent disclosure contemplates using spontaneous mutants ofphotosynthetic microorganisms that demonstrate a growth advantage undera defined growth condition. Among other benefits, the ability to producelarge amounts of triglycerides from minimal energy and nutrient inputmakes the modified photosynthetic microorganism, e.g., Cyanobacteria, ofthe present disclosure a readily manageable and efficient source offeedstock in the subsequent production of both biofuels, such asbiodiesel, as well as specialty chemicals, such as glycerin.

Methods of Producing Modified Photosynthetic Microorganisms

Embodiments of the present disclosure also include methods of producingthe modified photosynthetic microorganisms (e.g., Cyanobacterium)described herein.

In certain embodiments, the present disclosure comprises methods ofmodifying a photosynthetic microorganism to produce a modifiedphotosynthetic microorganism that produces an increased amount oflipids, e.g., triglycerides, relative to a corresponding wild typephotosynthetic microorganism or a differently modified photosyntheticmicroorganism (e.g., one that expresses DGAT but not a form thatselectively localizes to an intracellular region such as a membrane,including the plasma membrane), comprising introducing into themicroorganism one or more polynucleotides encoding a intracellularlocalization domain-DGAT fusion protein described herein, includingactive fragments or variants thereof.

Also included are methods of modifying a photosynthetic microorganism toproduce a modified photosynthetic microorganism that has improved cellgrowth characteristics, relative to a corresponding, DGAT-expressingmodified photosynthetic microorganism where the DGAT does not have aheterologous intracellular localization domain (e.g., a wild-type DGAT),comprising introducing into the microorganism one or morepolynucleotides encoding a intracellular localization domain-DGAT fusionprotein described herein, including active fragments or variantsthereof.

The methods may further comprise a step of selecting for photosyntheticmicroorganisms in which the one or more desired polynucleotides weresuccessfully introduced, where the polynucleotides were, e.g., presentin a vector that expressed a selectable marker, such as an antibioticresistance gene. As one example, selection and isolation may include theuse of antibiotic resistant markers known in the art (e.g., kanamycin,spectinomycin, and streptomycin).

In certain aspects, such photosynthetic microorganisms can be furthermodified by increasing the expression of one or more lipid biosynthesisproteins, for instance, by introducing an exogenous copy of apolynucleotide that encodes a lipid biosynthesis protein, by increasingexpression of an endogenous lipid biosynthesis protein, or both. In someaspects, such photosynthetic microorganisms can be further modified byincreasing the expression of one or more proteins associated withglycogen breakdown, for instance, by introducing an exogenous copy of apolynucleotide that encodes a glycogen breakdown protein, by increasingexpression of an endogenous glycogen breakdown protein, or both.

Thus, in certain embodiments, the present disclosure includes methods ofproducing a modified photosynthetic microorganism, e.g., aCyanobacteria, comprising: (1) introducing into the photosyntheticmicroorganism one or more polynucleotides encoding one or moreintracellular localization domain-DGAT fusion proteins, and (2)introducing into the photosynthetic microorganism one or moreoperatively linked promoters (e.g., inducible or regulable promoters)into a region upstream of an endogenous lipid biosynthesis proteincoding sequence, and/or introducing one or more polynucleotides encodinga lipid biosynthesis protein, or a fragment or variant thereof.Exemplary lipid biosynthesis proteins include any one or more of acylcarrier proteins (ACP), acyl ACP synthetases (Aas), acyl-ACP reductases,alcohol dehydrogenases, aldehyde dehydrogenases, aldehydedecarbonylases, thioesterases (TES), acetyl coenzyme A carboxylases(ACCase), phosphatidic acid phosphatases (PAP; or phosphatidatephosphatases), triacylglycerol (TAG) hydrolases, fatty acyl-CoAsynthetases, and lipases/phospholipases, including any combinationthereof.

Certain embodiments include methods of producing a modifiedphotosynthetic microorganism, e.g., a Cyanobacteria, comprising: (1)introducing into the photosynthetic microorganism one or morepolynucleotides encoding one or more intracellular localizationdomain-DGAT fusion proteins, and (2) introducing into the photosyntheticmicroorganism one or more operatively linked promoters (e.g., inducibleor regulable promoters) into a region upstream of an endogenous glycogenbreakdown protein coding sequence, and/or introducing one or morepolynucleotides encoding a glycogen breakdown protein, or a fragment orvariant thereof. Exemplary glycogen breakdown proteins include any oneor more of glycogen phosphorylase (GlgP), glycogen isoamylase (GlgX),glucanotransferase (MalQ), phosphoglucomutase (Pgm), glucokinase (Glk),and/or phosphoglucose isomerase (Pgi), including any combinationthereof.

Particular embodiments include methods of producing a modifiedphotosynthetic microorganism, e.g., a Cyanobacteria, comprising: (1)Introducing into the photosynthetic microorganism one or morepolynucleotides encoding one or more intracellular localizationdomain-DGAT fusion proteins, (2) introducing into the photosyntheticmicroorganism one or more operatively linked promoters (e.g., inducibleor regulable promoters) into a region upstream of an endogenous lipidbiosynthesis protein coding sequence, and/or introducing one or morepolynucleotides encoding a lipid biosynthesis protein, or a fragment orvariant thereof, and (3) introducing into the photosyntheticmicroorganism one or more operatively linked promoters (e.g., inducibleor regulable promoters) into a region upstream of an endogenous glycogenbreakdown protein coding sequence, and/or introducing one or morepolynucleotides encoding a glycogen breakdown protein, or a fragment orvariant thereof.

In particular embodiments, the lipid biosynthesis protein is an acylcarrier protein (ACP), an acyl-ACP synthetase (Aas), or both. Forinstance, certain embodiments include methods for producing a modifiedphotosynthetic microorganism, e.g., a Cyanobacteria, comprising: (1)introducing into the photosynthetic microorganism one or morepolynucleotides encoding one or more intracellular localizationdomain-DGAT fusion proteins, (2) introducing into the photosyntheticmicroorganism one or more operatively linked promoters (e.g., inducibleor regulable promoters) into a region upstream of an endogenous ACPcoding sequence, and/or introducing one or more polynucleotides encodingan ACP, or a fragment or variant thereof. These and related methods canfurther comprise (3) introducing into the photosynthetic microorganismone or more operatively linked promoters (e.g., inducible or regulablepromoters) into a region upstream of an endogenous TES, ACCase, TAGhydrolase, fatty acyl CoA synthetase, PAP, and/or phospholipase codingsequence, and/or Introducing one or more polynucleotides encoding TES,ACCase, TAG hydrolase, fatty acyl CoA synthetase, PAP, and/orphospholipase, or a fragment or variant thereof.

Some embodiments include methods for producing a modified photosyntheticmicroorganism, e.g., a Cyanobacteria, comprising: (1) introducing intothe photosynthetic microorganism one or more polynucleotides encodingone or more intracellular localization domain-DGAT fusion proteins, (2)introducing into the photosynthetic microorganism one or moreoperatively linked promoters (e.g., inducible or regulable promoters)into a region upstream of an endogenous Aas coding sequence, and/orintroducing one or more polynucleotides encoding an Aas polypeptide, ora fragment or variant thereof. These and related methods can furthercomprise (3) introducing into the photosynthetic microorganism one ormore operatively linked promoters (e.g., inducible or regulablepromoters) into a region upstream of an endogenous TES, ACCase, TAGhydrolase, fatty acyl CoA synthetase, PAP, and/or phospholipase codingsequence, and/or introducing one or more polynucleotides encoding TES,ACCase, TAG hydrolase, fatty acyl CoA synthetase, PAP, and/orphospholipase, or a fragment or variant thereof.

Certain embodiments include methods of producing a modifiedphotosynthetic microorganism, e.g., a Cyanobacteria, comprising: (1)Introducing into the photosynthetic microorganism one or morepolynucleotides encoding one or more intracellular localizationdomain-DGAT fusion proteins, (2) introducing into the photosyntheticmicroorganism one or more operatively linked promoters (e.g., inducibleor regulable promoters) into a region upstream of an endogenous ACPcoding sequence, and/or introducing one or more polynucleotides encodingan ACP, or a fragment or variant thereof, and (3) Introducing into thephotosynthetic microorganism one or more operatively linked promoters(e.g., inducible or regulable promoters) into a region upstream of anendogenous Aas coding sequence, and/or introducing one or morepolynucleotides encoding an Aas polypeptide, or a fragment or variantthereof. These and related methods can further comprise (4) introducinginto the photosynthetic microorganism one or more operatively linkedpromoters (e.g., inducible or regulable promoters) into a regionupstream of an endogenous TES, ACCase, TAG hydrolase, fatty acyl CoAsynthetase, PAP, and/or phospholipase coding sequence, and/orintroducing one or more polynucleotides encoding TES, ACCase, TAGhydrolase, fatty acyl CoA synthetase, PAP, and/or phospholipase, or afragment or variant thereof.

In some embodiments, the lipid biosynthesis protein is an acyl-ACPreductase, optionally in combination with an overexpressed alcoholdehydrogenase, for instance, to increase production of triglyceridesand/or produce wax esters. Certain embodiments thus include methods ofproducing a modified photosynthetic microorganism, e.g., aCyanobacteria, comprising: (1) introducing into the photosyntheticmicroorganism one or more polynucleotides encoding one or moreintracellular localization domain-DGAT fusion proteins, and (2)introducing into the photosynthetic microorganism one or moreoperatively linked promoters (e.g., inducible or regulable promoters)into a region upstream of an endogenous acyl-ACP reductase codingsequence, and/or introducing one or more polynucleotides encoding anacyl-ACP reductase, or a fragment or variant thereof.

For wax ester production, also included are methods of producing amodified photosynthetic microorganism, e.g., a Cyanobacteria,comprising: (1) introducing into the photosynthetic microorganism one ormore polynucleotides encoding one or more intracellular localizationdomain-DGAT fusion proteins, (2) introducing into the photosyntheticmicroorganism one or more operatively linked promoters (e.g., inducibleor regulable promoters) into a region upstream of an endogenous acyl-ACPreductase coding sequence, and/or introducing one or morepolynucleotides encoding an acyl-ACP reductase, or a fragment or variantthereof, and (3) introducing into the photosynthetic microorganism oneor more operatively linked promoters (e.g., inducible or regulablepromoters) into a region upstream of an endogenous alcohol dehydrogenasecoding sequence, and/or introducing one or more polynucleotides encodingan alcohol dehydrogenase, or a fragment or variant thereof.

Any of the photosynthetic microorganisms described herein can be furthermodified by reducing expression and/or activity of one or moreendogenous genes/proteins associated with glycogen synthesis and/orstorage, one or more endogenous aldehyde dehydrogenases, one or moreendogenous aldehyde decarbonylases, and/or one or more endogenous Aaspolypeptides. Exemplary genes/proteins associated with glycogensynthesis and/or storage include glgA, glgC, and pgm.

In particular embodiments, expression or activity is reduced by knockingout or knocking down one or more alleles of the one or more genes. Inparticular embodiments, expression or activity of the one or more genesis reduced by contacting the photosynthetic microorganism with anantisense oligonucleotide or interfering RNA, e.g., an siRNA, thattargets the one or more genes. In certain embodiments, a vector thatexpresses a polynucleotide that hybridizes to the one or more genes,e.g., an antisense oligonucleotide or an siRNA is introduced into thephotosynthetic microorganism. Also included is the generation ofmutants, such as point mutants, insertions, or full or partial deletionsof a gene of interest and/or one or more of its regulatory elements(e.g., promoters, enhancers), to reduce expression and/or activity of aprotein of interest. Natural selection or directed selection can also beused to identify naturally-occurring mutants having reduced expressionand/or activity of a protein of interest.

For instance, particular embodiments include methods for producing amodified photosynthetic microorganism having reduced expression and/oractivity of an aldehyde dehydrogenase, an aldehyde decarbonylase, orboth. These and related embodiments may comprise (1) introducing intothe photosynthetic microorganism one or more polynucleotides encodingone or more intracellular localization domain-DGAT fusion proteins, and(2) introducing one or more mutations into an endogenous gene encodingan aldehyde dehydrogenase, such as a point mutation, insertion, or fullor partial deletion, which reduces expression and/or activity of thealdehyde dehydrogenase, e.g., renders the aldehyde dehydrogenase“non-functional,” as described herein. Also included are methods forproducing a modified photosynthetic microorganism, comprising (1)introducing into the photosynthetic microorganism one or morepolynucleotides encoding one or more intracellular localizationdomain-DGAT fusion proteins, and (2) introducing one or more mutationsinto an endogenous gene encoding an aldehyde decarbonylase, such as apoint mutation, insertion, or full or partial deletion, which reducesexpression and/or activity of the aldehyde decarbonylase.

Some embodiments include methods for producing a modified photosyntheticmicroorganism, comprising (1) introducing into the photosyntheticmicroorganism one or more polynucleotides encoding one or moreintracellular localization domain-DGAT fusion proteins, (2) introducingone or more mutations into an endogenous gene encoding an aldehydedehydrogenase, such as a point mutation, insertion, or full or partialdeletion, which reduces expression and/or activity of the aldehydedehydrogenase, and (3) introducing one or more mutations into anendogenous gene encoding an aldehyde decarbonylase, such as a pointmutation, insertion, or full or partial deletion, which reducesexpression and/or activity of the aldehyde decarbonylase.

Particular methods include producing a modified photosyntheticmicroorganism having increased expression of an acyl-ACP reductase andan alcohol dehydrogenase, in combination with reduced expression and/oractivity of an aldehyde dehydrogenase, reduced expression and/oractivity of an aldehyde decarbonylase, or both. These and relatedembodiments can be useful in the production of wax esters, as describedherein. Some embodiments thus include methods for producing a modifiedphotosynthetic microorganism, comprising (1) introducing into thephotosynthetic microorganism one or more polynucleotides encoding one ormore intracellular localization domain-DGAT fusion proteins, (2)introducing into the photosynthetic microorganism one or moreoperatively linked promoters (e.g., inducible or regulable promoters)into a region upstream of an endogenous acyl-ACP reductase codingsequence, and/or introducing one or more polynucleotides encoding anacyl-ACP reductase, or a fragment or variant thereof, (3) introducinginto the photosynthetic microorganism one or more operatively linkedpromoters (e.g., inducible or regulable promoters) into a regionupstream of an endogenous alcohol dehydrogenase coding sequence, and/orintroducing one or more polynucleotides encoding an alcoholdehydrogenase, or a fragment or variant thereof, and either or both of(4) introducing one or more mutations into an endogenous gene encodingan aldehyde dehydrogenase, such as a point mutation, insertion, or fullor partial deletion, which reduces expression and/or activity of thealdehyde dehydrogenase, and (5) introducing one or more mutations intoan endogenous gene encoding an aldehyde decarbonylase, such as a pointmutation, insertion, or full or partial deletion, which reducesexpression and/or activity of the aldehyde decarbonylase. In certainembodiments, for instance, where the photosynthetic microorganism is S.elongatus, the aldehyde dehydrogenase is encoded by orf0489, and thealdehyde decarbonylase is encoded by orf1593.

Photosynthetic microorganisms, e.g., Cyanobacteria, may be geneticallymodified according to techniques known in the art, e.g., delete aportion or all of a gene or to introduce a polynucleotide that expressesa functional polypeptide. As noted above, in certain aspects, geneticmanipulation in photosynthetic microorganisms, e.g., Cyanobacteria, canbe performed by the introduction of non-replicating vectors whichcontain native photosynthetic microorganism sequences, exogenous genesof interest, and selectable markers or drug resistance genes. Uponintroduction into the photosynthetic microorganism, the vectors may beintegrated into the photosynthetic microorganism's genome throughhomologous recombination. In this way, an exogenous gene of interest andthe drug resistance gene are stably integrated into the photosyntheticmicroorganism's genome. Such recombinants cells can then be isolatedfrom non-recombinant cells by drug selection. Cell transformationmethods and selectable markers for Cyanobacteria are also well known inthe art (see, e.g., Wirth, Mol Gen Genet 216:175-7, 1989; andKoksharova, Appl Microbiol Biotechnol 58:123-37, 2002; and TheCyanobacteria: Molecular Biology, Genetics, and Evolution (eds. AntonioHerrera and Enrique Flores) Caister Academic Press, 2008, each of whichis incorporated by reference for their description on gene transfer intoCyanobacteria, and other information on Cyanobacteria).

In certain embodiments, an endogenous version of a protein (e.g., ACP,Aas, TES, ACCase, TAG hydrolase, fatty acyl-CoA synthetase, PAP, PL), ifpresent, can be overexpressed by introducing a heterologous or otherpromoter upstream of the endogenous gene encoding that protein, i.e.,the naturally-occurring version of that gene. Such promoters may beconstitutive or inducible.

Generation of deletions or mutations of any of the one or more genesassociated with the biosynthesis or storage of glycogen can beaccomplished according to a variety of methods known in the art,including the use of a non-replicating, selectable vector system that istargeted to the upstream and downstream flanking regions of a given gene(e.g., glgC, pgm), and which recombines with the Cyanobacterial genomeat those flanking regions to replace the endogenous coding sequence withthe vector sequence. Given the presence of a selectable marker in thevector sequence, such as a drug selectable marker, Cyanobacterial cellscontaining the gene deletion can be readily isolated, identified andcharacterized. Such selectable vector-based recombination methods neednot be limited to targeting upstream and downstream flanking regions,but may also be targeted to internal sequences within a given gene, aslong as that gene is rendered “non-functional,” as described herein.

The generation of deletions or mutations can also be accomplished usingantisense-based technology. For instance, Cyanobacteria have been shownto contain natural regulatory events that rely on antisense regulation,such as a 177-nt ncRNA that is transcribed in antisense to the centralportion of an iron-regulated transcript and blocks its accumulationthrough extensive base pairing (see, e.g., Dühring, et al., Proc. Natl.Acad. Sci. USA 103:7054-7058, 2006), as well as a alr1690 mRNA thatoverlaps with, and is complementary to, the complete furA gene, whichacts as an antisense RNA (α-furA RNA) interfering with furA transcripttranslation (see, e.g., Hernandez et al., Journal of Molecular Biology355:325-334, 2006). Thus, the incorporation of antisense moleculestargeted to genes involved in glycogen biosynthesis or storage would besimilarly expected to negatively regulate the expression of these genes,rendering them “non-functional,” as described herein.

As used herein, antisense molecules encompass both single anddouble-stranded polynucleotides comprising a strand having a sequencethat is complementary to a target coding strand of a gene or mRNA. Thus,antisense molecules include both single-stranded antisenseoligonucleotides and double-stranded siRNA molecules.

Other modifications described herein may be produced using standardprocedures and reagents, e.g., vectors, available in the art. Relatedmethods are described in PCT Application No. WO 2010/075440, which ishereby incorporated by reference in its entirety.

Methods of Producing Lipids

The modified photosynthetic microorganisms and methods of the presentdisclosure may be used to produce lipids, such as fatty acids,triglycerides, and/or wax esters. Accordingly, the present disclosureprovides methods of producing lipids, comprising culturing any of themodified photosynthetic microorganisms of the present disclosure(described elsewhere herein)

In one embodiment, the modified photosynthetic microorganism is aCyanobacterium that produces or accumulates increased lipids relative toan unmodified or wild-type Cyanobacterium of the same species, or adifferently modified Cyanobacterium of the same species. In certainembodiments, the modified photosynthetic microorganism is aCyanobacterium that produces or accumulates increased triglyceridesrelative to an unmodified or wild-type Cyanobacterium of the samespecies, or a differently modified Cyanobacterium of the same species.In certain instances, the differently modified Cyanobacterium expressesa wild-type DGAT, and no other form(s) of DGAT. Other examples ofdifferently modified Cyanobacteria are described herein. In certainaspects, increased triglyceride production is associated with improvedcell growth characteristics relative to the differently modifiedCyanobacterium, e.g., increased cell survival over time, and is thusmeasured over time, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, ormore days post-culture or post-induction of DGAT expression, or in acontinuous culture system.

In one embodiment, the modified photosynthetic microorganism is aCyanobacterium that produces or accumulates increased wax estersrelative to an unmodified or wild-type Cyanobacterium of the samespecies, or a differently modified Cyanobacterium of the same species.In these and related embodiments, the Cyanobacterium overexpresses anacyl-ACP reductase and an alcohol dehydrogenase, in combination with anintracellular localization domain-DGAT fusion protein. In someembodiments, the differently modified Cyanobacterium is one thatexpresses DGAT in combination with an acyl-ACP reductase and an alcoholdehydrogenase, and thus produces wax esters, but expresses a wild-typeDGAT and no other form of DGAT. In some aspects, increased wax esterproduction is associated with improved cell growth characteristicsrelative to the differently modified Cyanobacterium, e.g., increasedcell survival over time, and is thus measured over time, e.g., 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days post-culture orpost-induction of DGAT expression, or in a continuous culture system.

In certain embodiments, the one or more introduced polynucleotides arepresent in one or more expression constructs. In particular embodiments,the one or more expression constructs comprises one or more induciblepromoters. In certain embodiments, the one or more expression constructsare stably integrated into the genome of the modified photosyntheticmicroorganism.

In certain embodiments, the introduced polynucleotide encoding anintroduced protein is present in an expression construct comprising aweak promoter under non-induced conditions. In certain embodiments, oneor more of the introduced polynucleotides are codon-optimized forexpression in a Cyanobacterium, e.g., a Synechococcus elongatus.

In particular embodiments, the photosynthetic microorganism is aSynechococcus elongatus, such as Synechococcus elongatus strain PCC7942or a salt tolerant variant of Synechococcus elongatus strain PCC7942. Inparticular embodiments, the photosynthetic microorganism is aSynechococcus sp. PCC 7002 or a Synechocystis sp. PCC6803.

Photosynthetic microorganisms may be cultured according to techniquesknown in the art. For example, Cyanobacteria may be cultured orcultivated according to techniques known in the art, such as thosedescribed in Acreman et al. (Journal of Industrial Microbiology andBiotechnology 13:193-194, 1994), in addition to photobioreactor basedtechniques, such as those described in Nedbal et al. (Biotechnol Bioeng.100:902-10, 2008). One example of typical laboratory culture conditionsfor Cyanobacterium is growth in BG-11 medium (ATCC Medium 616) at 30° C.in a vented culture flask with constant agitation and constantillumination at 30-100 μmole photons m⁻² sec⁻¹.

A wide variety of mediums are available for culturing Cyanobacteria,including, for example, Aiba and Ogawa (AO) Medium, Allen and ArnonMedium plus Nitrate (ATCC Medium 1142), Antia's (ANT) Medium, AquilMedium, Ashbey's Nitrogen-free Agar, ASN-III Medium, ASP 2 Medium, ASWMedium (Artificial Seawater and derivatives), ATCC Medium 617 (BG-11 forMarine Blue-Green Algae; Modified ATCC Medium 616 [BG-11 medium]), ATCCMedium 819 (Blue-green Nitrogen-fixing Medium; ATCC Medium 616 [BG-11medium] without NO₃), ATCC Medium 854 (ATCC Medium 616 [BG-11 medium]with Vitamin B₁₂), ATCC Medium 1047 (ATCC Medium 957 [MN marine medium]with Vitamin B₁₂), ATCC Medium 1077 (Nitrogen-fixing marine medium; ATCCMedium 957 [MN marine medium] without NO₃), ATCC Medium 1234 (BG-11Uracil medium; ATCC Medium 616 [BG-11 medium] with uracil), BeggiatoaMedium (ATCC Medium 138), Beggiatoa Medium 2 (ATCC Medium 1193), BG-11Medium for Blue Green Algae (ATCC Medium 616), Blue-Green (BG) Medium,Bold's Basal (BB) Medium, Castenholtz D Medium, Castenholtz D MediumModified (Halophilic cyanobacteria), Castenholtz DG Medium, CastenholtzDGN Medium, Castenholtz ND Medium, Chloroflexus Broth, ChloroflexusMedium (ATCC Medium 920), Chu's #10 Medium (ATCC Medium 341), Chu's #10Medium Modified, Chu's #11 Medium Modified, DCM Medium, DYIV Medium, E27Medium, E31 Medium and Derivatives, f/2 Medium, f/2 Medium Derivatives,Fraquil Medium (Freshwater Trace Metal-Buffered Medium), Gorham's Mediumfor Algae (ATCC Medium 625), h/2 Medium, Jaworski's (JM) Medium, KMedium, L1 Medium and Derivatives, MN Marine Medium (ATCC Medium 957),Plymouth Erdschreiber (PE) Medium, Prochlorococcus PC Medium, ProteosePeptone (PP) Medium, Prov Medium, Prov Medium Derivatives, S77 plusVitamins Medium, 588 plus Vitamins Medium, Saltwater Nutrient Agar (SNA)Medium and Derivatives, SES Medium, SN Medium, Modified SN Medium, SNAXMedium, Soil/Water Biphasic (S/W) Medium and Derivatives, SOT Medium forSpirulina: ATCC Medium 1679, Spirulina (SP) Medium, van Rijn and Cohen(RC) Medium, Walsby's Medium, Yopp Medium, and Z8 Medium, among others.

In particular embodiments, the modified photosynthetic microorganismsare cultured under conditions suitable for inducing expression of theintroduced polynucleotide(s), e.g., wherein the introducedpolynucleotide(s) comprise an inducible promoter. Conditions andreagents suitable for inducing inducible promoters are known andavailable in the art. Also included are the use of auto-inductivesystems, for example, where a metabolite represses expression of theintroduced polynucleotide, and the use of that metabolite by themicroorganism over time decreases its concentration and thus itsrepressive activities, thereby allowing increased expression of thepolynucleotide sequence.

In certain embodiments, modified photosynthetic microorganisms, e.g.,Cyanobacteria, are grown under conditions favorable for producinglipids, triglycerides and/or fatty acids. In particular embodiments,light intensity is between 100 and 2000 uE/m2/s, or between 200 and 1000uE/m2/s. In particular embodiments, the pH range of culture media isbetween 7.0 and 10.0. In certain embodiments, CO₂ is injected into theculture apparatus to a level in the range of 1% to 10%. In particularembodiments, the range of CO₂ is between 2.5% and 5%. In certainembodiments, nutrient supplementation is performed during the linearphase of growth. Each of these conditions may be desirable fortriglyceride production.

In certain embodiments, the modified photosynthetic microorganisms arecultured, at least for some time, under static growth conditions asopposed to shaking conditions. For example, the modified photosyntheticmicroorganisms may be cultured under static conditions prior to Inducingexpression of an introduced polynucleotide (e.g., intracellularlocalization domain-DGAT fusion, acyl-ACP reductase, ACP, Aas, ACP/Aas,glycogen breakdown protein, ACCase, DGAT, fatty acyl-CoA synthetase,aldehyde dehydrogenase, alcohol dehydrogenase) and/or the modifiedphotosynthetic microorganism may be cultured under static conditionswhile expression of an introduced polynucleotide is being induced, orduring a portion of the time period during which expression of anintroduced polynucleotide is being induced. Static growth conditions maybe defined, for example, as growth without shaking or growth wherein thecells are shaken at less than or equal to 30 rpm or less than or equalto 50 rpm.

In certain embodiments, the modified photosynthetic microorganisms arecultured, at least for some time, in media supplemented with varyingamounts of bicarbonate. For example, the modified photosyntheticmicroorganisms may be cultured with bicarbonate at 5, 10, 20, 50, 75,100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 mM bicarbonate priorto inducing expression of an introduced polynucleotide (e.g.,membrane-targeting domain-DGAT fusion protein, acyl-ACP reductase,aldehyde dehydrogenase, ACP, Aas, ACP/Aas, glycogen breakdown protein,ACCase, DGAT, fatty acyl-CoA synthetase, alcohol dehydrogenase) and/orthe modified photosynthetic microorganism may be cultured withaforementioned bicarbonate concentrations while expression of anintroduced polynucleotide is being induced, or during a portion of thetime period during which expression of an introduced polynucleotide isbeing induced.

In related embodiments, modified photosynthetic organisms and methods ofthe present disclosure may be used in the production of a biofuel and/ora specialty chemical, such as glycerin or a wax ester. Thus, inparticular embodiments, a method of producing a biofuel comprisesculturing any of the modified photosynthetic microorganisms of thepresent disclosure under conditions wherein the modified photosyntheticmicroorganism accumulates an increased amount of total cellular lipid,fatty acid, wax ester, and/or triglyceride, as compared to acorresponding wild-type photosynthetic microorganism, obtaining cellularlipid, fatty acid, wax ester, and/or triglyceride from themicroorganism, and processing the obtained cellular lipid, fatty acid,wax ester, and/or triglyceride to produce a biofuel. In anotherembodiment, a method of producing a biofuel comprises processing lipids,fatty acids, wax esters, and/or triglycerides produced by a modifiedphotosynthetic microorganism of the present disclosure to produce abiofuel. In a further embodiment, a method of producing a biofuelcomprises obtaining lipid, fatty acid, wax esters, and/or triglycerideproduced by a modified photosynthetic microorganism of the presentinvention, and processing the obtained cellular lipid, fatty acid, waxester, and/or triglyceride to produce a biofuel. In particularembodiments, the modified photosynthetic organism is grown underconditions wherein it has reduced growth but maintains photosynthesis.

Methods of processing lipids from microorganisms to produce a biofuel orspecialty chemical, e.g., biodiesel, are known and available in the art.For example, triglycerides may be transesterified to produce biodiesel.Transesterification may be carried out by any one of the methods knownin the art, such as alkali-, acid-, or lipase-catalysis (see, e.g.,Singh et al., Recent Pat Biotechnol. 2008, 2(2):130-143). Variousmethods of transesterification utilize, for example, use of a batchreactor, a supercritical alcohol, an ultrasonic reactor, or microwaveirradiation (Such methods are described, e.g., in Jeong and Park, ApplBiochem Biotechnol. 2006, 131(1-3):668-679; Fukuda et al., Journal ofBioscience and Engineering. 2001, 92(5):405-416; Shah and Gupta,Chemistry Central Journal. 2008, 2(1):1-9; and Carrillo-Munoz et al., JOrg Chem. 1996, 61(22):7746-7749). The biodiesel may be furtherprocessed or purified, e.g., by distillation, and/or a biodieselstabilizer may be added to the biodiesel, as described in U.S. PatentApplication Publication No. 2008/0282606.

Certain embodiments of the present disclosure now will be illustrated bythe following Examples. The present disclosure may, however, be embodiedin many different forms and should not be construed as limited to theembodiments set forth herein; rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the disclosure to those skilled in the art.

EXAMPLES Example 1

FIGS. 1A and 1B show a measurement of phycobilisomes, comparingwild-type to NbIA overexpressor. Synechococcus PCC7942 wild type,PCC7942 with an exogenous NbIA gene, uninduced or PCC7942 with anexogenous NbIA gene, induced. Samples were collected at time zero (FIG.1A) and at six hours (FIG. 1B) after induction of the NbIA gene. Wholecells were examined by spectrophotometry, and absorbance as a functionof wavelength was determined. The three major peaks represent absorptionby chlorophyll A (at approximately 420 and 680 nm) and byphycobiliprotein (at approximately 630 nm). Induction of NbIA caused arapid decrease in light absorption by phycobiliprotein. Some reductionin the 680 nm chlorophyll A peak was also observed. These data show thatphycobilisomes are reduced after modulated NbIA expression, indicatingthat modulating NbIA expression reduce phycobilisome abundance.

Example 2

Normalized photosynthetic activities of suspensions of wild type andmodified cyanobacteria containing an arabinose-induced over-expressionsystem of the gene nblA. Triplicate cultures of wild-type Synechococcussp. PCC 7942, pBAD nblA uninduced and pBAD nblA induced (with 0.02%arabinose added) were harvested in log-phase (between OD₇₅₀ values of0.4 and 0.6) and re-suspended in BG-11 medium with 20 mM PotassiumPhosphate (pH 7.5) and 10 mM Sodium Bicarbonate additions. Thesesuspensions were Illuminated with Red+Blue LEDs in a calibrated WalzDual Pam 100 Fluorometer (Walz, Germany) to total light Intensitiesbetween 0 and 600 μE*m⁻²*s⁻¹ and oxygen concentration was monitored by aNeoFox Oxygen Sensor (Ocean Optics, USA) continuously every second for120 seconds at each light intensity. The slopes of the linear O₂production rate was then found and plotted above for each culture time(n=3 for each type).

As shown in FIG. 2, oxygen evolution rates of suspensions ofcyanobacteria Synechococcus sp. PCC 7942 (herein denoted S7942)overexpressing nblA are higher than those of wild-type and an uninducedcontrol strain (pBAD nblA uninduced) at incident light intensitiesbetween 100 and 600 PE*m⁻²*s⁻¹. This is due to a decreased absorbance inwhole cell spectra in the wavelength region of phycobilisomes as shownin FIG. 3. Whole cells were examined by spectrophotometry, andabsorbance as a function of wavelength was determined. The decrease inabsorbance by phycobiliprotein (at approximately 630 nm) as in FIG. 1 isshown. Induction of NbIA caused a rapid decrease in light absorption byphycobiliprotein.

Example 3

Cultures of a control strain (wild type Synechococcus sp. PCC7942) inthe presence of 0.02% arabinose (which does not change opticalcharacteristics in presence of arabinose), pBAD nblA, and pBAD nblA inthe presence of 0.02% arabinose were grown in triplicate cultures at 30degrees Celsius in photobioreactors (Phenometrics, USA), top lit with2500 μE*m⁻¹*s⁻¹ incident white light LEDs with bubbling of 2% CO₂ inair. The medium used was BG-11+10 mM sodium phosphate (pH 7.1)+5 μg/mLKanamycin (which all strains had resistance markers for). Strains withoverexpression of nblA by induction with arabinose grew better than bothcontrols as measured by optical density (OD 750 nm) and dry weight(normalized to culture volume) as shown in FIGS. 4 A and B,respectively.

Example 4

NY001, a strain of PCC7942 with a native copy of nblA gene behind itsnative promoter plus a second copy of nblA gene behind an arabinoseinducible promoter (pBAD) has increased expression of total nblA geneexpression relative to wild type as shown by q-RT-PCR. Also, NY016, astrain of PCC7942 with a native copy of the nblA gene behind its nativepromoter, plus a second copy of nblA gene behind a constitutivehigh-expression promoter (pSYNPCC7942_1306) has a similar enhancement intotal nblA gene expression. These observations are via quantitativereverse transcriptase polymerase chain reaction (q-RT-PCR) as shown inFIG. 5. For this experiment, triplicate cultures grown under moderatewhite light from cool-white fluorescent bulbs (light intensityapproximately 120 μE*m⁻²*s⁻¹) at 30 degrees Celsius in BG-11 mediasupplemented with 20 mM sodium phosphate (pH 7.1) and 0.02% L-arabinosefor NY001_1. Samples from each culture were harvested in mid-log phase;cells were pelleted by centrifugation at 22,000×g for 5 minutes andsupernatant was discarded. RNA was extracted from the remaining cellpellets and used to generate a cDNA library using a Qiagen Rneasy MiniKit and the Rnase-free Dnase Set (Qiagen, USA). This cDNA library wasused for q-RT-PCR, which was run relative to the rnpB housekeeping gene.Resulting relative expression levels are then shown as log₂(fold change)of desired gene (in this case nblA) relative to a control (in this casewild-type (“WT”)).

Also, NY016 has increased photosynthetic activity, comparable to pBADinduced nblA (NY001_1) as measured by oxygen evolution rates shown inFIG. 6. Normalized photosynthetic activities of suspensions of wild typeand NY016 were measured from triplicate cultures. Cells were harvestedin log-phase (between OD₇₅₀ values of 0.4 and 0.6) and re-suspended inBG-11 medium with 20 mM Potassium Phosphate (pH 7.5) and 10 mM SodiumBicarbonate additions. These suspensions were illuminated with Red+BlueLEDs in a calibrated Walz Dual Pam 100 Fluorometer (Walz, Germany) tototal light intensities between 0 and 1000 μE*m⁻²*s⁻¹ and oxygenconcentration was monitored by a NeoFox Oxygen Sensor (Ocean Optics,USA) continuously every second for 120 seconds at each light intensity.The slope of the linear O₂ production rate was then found and plottedabove for each culture time (n=3 for each type).

NY016 also grows faster and to higher densities than the control strainNY048 (containing only the native copy of nblA behind its nativepromoter; It is a wild-type like strain with an added cassette forantibiotic resistance only) as shown in FIG. 7. Here, Cultures of acontrol strain (which contains only native nblA gene) and NY016 (whichcontains the native nblA gene plus a second, overexpressed copy of thenblA gene) were grown in triplicate cultures at 30 degrees Celsius inphotobioreactors (Phenometrics, USA), top lit with 2500 μE*m⁻²*s⁻¹incident white light LEDs with bubbling of 2% CO₂ in air. The mediumused was BG-11+10 mM sodium phosphate (pH 7.1)+2 μg/mL spectinomycin+2μg/mL streptomycin (which both NY016 and NY048 have resistance markersfor). NY016 grew better than control (NY048) as measured by opticaldensity (OD 750 nm) and dry weight (normalized to culture volume) asshown in FIGS. 7A and 7B, respectively. Whole-cell spectra were takenwith a spectrophotometer of aliquot samples from these reactors part waythrough the experiment (between day 2 and day 3) to verify thatdecreased absorbance was seen at and around 630 nm due to decreases inphycobilins from these samples. Representative normalized spectra areshown in FIG. 8 from these samples.

Example 5

Screening of natural mutants in populations of wild-type Synechococcussp. PCC 7942 for resistance to increased light was achieved by killingcultures of S7942 with high light treatment (>3000 μE*m⁻²*s⁻¹ white LEDlight) and plating survivors on agar plates. To achieve killing,cultures of wild-type presumed to contain a natural sub-population ofmutants were grown at 30 degrees Celcius to log-phase (approximately 0.5OD₇₅₀) and resuspended at 0.1 OD₇₅₀ in 50 mL volumes in glass bottles.The glass bottles were covered on one side with aluminum foil to reflectincoming light. The bottles were placed in a transparent water bathwhich was up against a panel of white LEDs. The LEDs were turned on for1 hour while samples in glass bottles were bubbled with air foragitation. The LED panels were turned off for 1 hour and then a secondhigh-light treatment was achieved by exposing the same samples to the onLEDs. Three rounds of high light exposure in total were performed. Theremaining culture after three rounds was spread on agar plates and leftto grow under moderate (approximately 100 μE*m⁻²*s⁻¹ light from coolwhite fluorescent bulbs). Cells that grew with discoloration wereconsidered mutants. At least three mutants isolated when cultured andtreated by light have a lower fraction of cell death induced by highlight treatment as shown in FIG. 9. For FIG. 9, high light treatmentswere performed on WT, and mutants isolated from the procedure asdescribed above. This procedure was repeated for WT, and mutants DC1,DC3 and DC4, and glass bottles were sampled for plating of smallaliquots before and after each round of high light exposure to followcell-death by high light. FIG. 9 shows that WT cell viability (asmeasured by Colony forming units (CFUs) per mL of culture in the glassbottles drops from 10⁷ to about 10² while mutants have higher CFUs after3 rounds of killing. Two of these mutants, DC1 and DC4 have decreasedabsorbances in pigment regions relative to WT as shown in FIG. 10, whichshows a normalized whole-cell spectrum taken as from whole-cellsuspensions of actively growing cultures of the strains. DC1 was shownto have an improved normalized oxygen evolution rate response to lightthan WT and DC4 as shown in FIG. 11. For this experiment, triplicatecultures of wild-type S7942, and mutants DC1 and DC4 were harvested inlog-phase (between OD₇₅₀ values of 0.4 and 0.6) and re-suspended inBG-11 medium with 20 mM Potassium Phosphate (pH 7.5) and 10 mM SodiumBicarbonate additions. These suspensions were illuminated with Red+BlueLEDs in a calibrated Walz Dual Pam 100 Fluorometer (Walz, Germany) tototal light intensities between 0 and 900 μE*m⁻²*s⁻¹ and oxygenconcentration was monitored by a NeoFox Oxygen Sensor (Ocean Optics,USA) continuously every second for 120 seconds at each light intensity.The slopes of the linear O₂ production rate was then found and plottedabove for each culture time (n=3 for each type).

Example 6

Screening of natural mutants in populations of wild-type Synechococcussp. PCC 7942 for resistance to metronidazole was achieved by killingcultures of S7942 with metronidazole treatment and plating survivors onagar plates. Wild-type cells presumed to contain a naturalsub-population of mutants were grown in BG11, suspended at a cellconcentration of 1×10⁶ cells/mL and treated with 4 mM metronidazole for1-2 hours under moderate light from cool white fluorescence bulbs at alight intensity of approximately 200 μmoles photons m⁻² s⁻¹. Theresulting culture was plated on agar plates incubated at a lightintensity of 100 μmoles photons m⁻² s⁻¹ to grow survivors. At leastseven mutants isolated from single colonies of the plates from thescreen when again cultured and treated with metronidazole have a lowerfraction of cell death induced by metranidazole treatment as shown inFIG. 12. Here, cultures of the WT and metronidazole (MZ) resistantmutants (1×10⁶ cells/mL) were Incubated with 4 mM MZ for 0, 1 and 2hours. Five μL of culture (˜5000 cells) were spotted from each flask andgrown on BG-11 plate at a light intensity of 100 μmoles photons m⁻² s⁻¹.

Example 7

NY056 [a strain of PCC7942 having a markerless deletion of nblA strain(NY052) with nblA behind pTrc added to neutral site 4] were grown inBG11+20 mM NaPi+0, 5, 20, 40, 60, 80, 100, or 120 uMisopropyl-β-D-1-thiogalactopyranoside (IPTG) (singlet cultures).Cultures were inoculated from log-phase culture of NY056 (no IPTG) at0.05 OD after 20 hours, OD750 values were as plotted as shown in FIG.13. The spectra of these cultures normalized to A800 were as shown inFIG. 14.

Two trends were noted. First, a strong trend of bilin decrease and,second, a weaker (with respect to IPTG addition) trend of chlorophylldecreases at 680 nm (but not 420 nm).

After ˜30 hours growth, O₂ evolution curves in the WALZ dual PAM 100were observed for cells resuspended at ˜2.0 OD750 in BG11+20 mM NaPi+10mM NaHCO₃. The curves are plotted and shown in FIG. 15. As shown in FIG.15, there is a dramatic, nearly 2-fold increase in O₂ evolution forcultures in 20 uM IPTG versus control (0 IPTG).

To better visualize the trend of Oz evolution vs IPTG added for growth,just the maximum light O₂ evolution for each culture at varying IPTG wasplotted as shown in FIG. 16. FIG. 16 shows a striking relationshipbetween bilin decrease and O₂ evolution increase versus chlorophylldecrease and O₂ gradual decrease.

The experiment discussed above was repeated with NY056 [the markerlessdeletion of nblA strain (NY052) with nblA behind pTrc added to neutralsite 4] in BG11+20 mM NaPi+0, 5, 10, 20, and 30 uM IPTG (singletcultures). This time, a range of bilin decreases was seen as shown bythe A800-normalized spectra of cultures grown for 30+ hrs in BG11+20 mMNaPi+0, 5, 10, 20, and 30 uM IPTG (See FIG. 17). In addition, 30 uMIPTG. 3 OD*mL worth of cells was also collected for SDS-PAGE analysis,which were flash frozen in 20 mM HEPES+10 mM EDTA+100 mM DTT+100 mMNa2CO3. After ˜30 hours growth, O₂ evolution curves in the WALZ dual PAM100 were observed for cells resuspended at ˜2.0 OD750 in BG11+20 mMNaPi+10 mM NaHCO₃. The curves are plotted and shown in FIG. 18. Themaximum light O₂ evolution for each culture at varying IPTG was plottedas shown in FIG. 19.

FIG. 20 shows the maximum light O₂ for both experiments combined in onemaximum light O₂ graph. FIG. 20 shows that the improvement in oxygenproduction (photosynthetic activity) varies as a function of the amountof NbIA expressed (ie the amount of IPTG inducer). An optimumimprovement is observed at about 20 micromolar IPTG. This corresponds toan approximately 40% decrease in light harvesting protein.

We claim:
 1. A cell culture comprising a Cyanobacteria cell population,wherein the Cyanobacteria cell population comprises genetically modifiedCyanobacteria cells that 1) are mutated to disrupt or delete one or moregenes encoding one or more light harvesting proteins; and 2) comprise anucleic acid sequence comprising an exogenous gene encoding an exogenouspolypeptide, and wherein when the Cyanobacteria cell culture is culturedunder light intensities between about a) 200 micromol photons per squaremeter per second; and b) about 1000 micromol photons per square meterper second, and the genetically modified Cyanobacteria cell populationexpresses the exogenous polypeptide.
 2. The cell culture of claim 1,wherein the modified Cyanobacteria cell population comprisesCyanobacteria cells that are genetically modified to delete aphycobilisome gene.
 3. The cell culture of claim 2, wherein thephycobilisome gene is a phycobiliprotein gene.
 4. The cell culture ofclaim 3, wherein the phycobiliprotein gene is a phycocyanin gene.
 5. Thecell culture of claim 1, wherein the modified Cyanobacteria cellcomprises addition of an endogenous NbIA gene.
 6. The cell culture ofclaim 1, wherein the exogenous polypeptide is a therapeutic polypeptide.7. The cell culture of claim 1, wherein the Cyanobacteria cellpopulation has 1) an increased biomass; and 2) an increased level ofphotosynthetic activity as compared to a corresponding unmodifiedCyanobacteria cell population.
 8. The cell culture of claim 1, whereinthe nucleic acid sequence comprising an exogenous gene encoding anexogenous polypeptide is present on a vector.
 9. The cell culture ofclaim 1, wherein the nucleic acid sequence comprising an exogenous geneencoding an exogenous polypeptide is integrated into the Spirulinagenome.
 10. A method of producing an exogenous polypeptide in aCyanobacteria culture comprising: 1) providing a modified Cyanobacteriacell population that comprises genetically modified Cyanobacteria cellsthat a) are mutated to disrupt or delete one or more genes encoding oneor more light harvesting proteins; and b) comprise a nucleic acidsequence comprising an exogenous gene encoding an exogenous polypeptide;2) growing the modified Cyanobacteria cell population wherein when theCyanobacteria cell culture is cultured under light intensities betweenabout a) 200 micromol photons per square meter per second; and b) about1000 micromol photons per square meter per second; growing the modifiedCyanobacteria cell population to obtain a Cyanobacteria culture; and,wherein the genetically modified Cyanobacteria cell population expressesthe exogenous polypeptide.
 11. The method of claim 1, wherein themodified Cyanobacteria cell population comprises Cyanobacteria cellsthat are genetically modified to delete a phycobilisome gene.
 12. Themethod of claim 11, wherein the phycobilisome gene is a phycobiliproteingene.
 13. The method of claim 12 wherein the phycobiliprotein gene is aphycocyanin gene.
 14. The method of claim 1, wherein the modifiedCyanobacteria cell comprises addition of an endogenous NbIA gene. 15.The method of claim 1, wherein the exogenous polypeptide is atherapeutic polypeptide.
 16. The method of claim 1, wherein theCyanobacteria cell population has 1) an increased biomass; and 2) anincreased level of photosynthetic activity as compared to acorresponding unmodified Cyanobacteria cell population.
 17. The methodof claim 1 wherein the nucleic acid sequence comprising an exogenousgene encoding an exogenous polypeptide is present on a vector.
 18. Themethod of claim 1, wherein the nucleic acid sequence comprising anexogenous gene encoding an exogenous polypeptide is integrated into theSpirulina genome.